Variants of human taste receptor genes

ABSTRACT

Identified herein are different forms of bitter receptor genes that occur in different humans. These alleles are generated by numerous coding single nucleotide polymorphisms (cSNP&#39;s) that occur within the members of the T2R gene family. Some SNP&#39;s cause amino acid substitutions, while others introduce chain termination codons, rendering the allele non-functional. Differences in these genes are believed to have a large effect on those individuals&#39; sense of bitter taste, such that these individuals perceive the taste of bitter substances differently than the rest of the population. The ability to assay this allelic information is useful in the development of flavorings and flavor enhancers, as it can be used to define large groups and populations who perceive bitter tastes differently. This in turn allows the taste preferences of these groups to be addressed at the molecular level for the first time.

CROSS REFERENCE TO RELATED APPLICATION

This is a §371 U.S. national stage of PCT/US2004/019489, filed Jun. 18,2004, which was published in English under PCT Article 2(2), and claimsthe benefit of U.S. Provisional Patent Application 60/480,035, filedJun. 19, 2003. Both applications are incorporated herein in theirentirety.

The Sequence Listing is provided in electronic format only on compactdiscs, as permitted under 37 CFR 1.52(e) and 1.821(c). The disc entitled“Sequ” contains the following file:

File name Size (KB) Date recorded onto disc 66168-03 Sequence 980 KBDec. 19, 2005 Listing.txt

FIELD

This disclosure relates to the field of taste reception, and moreparticularly to variations in taste receptors, such as bitter tastereceptors including those in the T2R family. It further relates tomethods for identifying individuals and populations having certain tastereceptor variants, and identifying compounds that interact with tastereceptors, including compounds that interact differentially withdifferent variants of a taste receptor.

BACKGROUND

Bitter taste is believed to have evolved in order to allow organisms todetect and avoid toxins from the environment. The sense of bitter tasteis mediated by a group of 24 apparently functional bitter taste receptorproteins that reside on the surface of taste cells within the taste budsof the tongue. These receptors are 7-transmembrane domain, G proteincoupled receptors, encoded by members of the T2R gene family. Incontrast to T1Rs, which also belong to the superfamily of Gprotein-coupled receptors and have a large N-terminal domain, T2R bittertaste receptors generally have a short extracellular N terminus. Thesecell surface receptors interact with tastants and initiate signalingcascades that culminate in neurotransmitter release and bitter tasteperception. The human genome contains 24 apparently functional T2Rgenes, which reside in three locations. Fourteen genes reside in acluster on chromosome 12p13, nine genes reside in a cluster onchromosome 7q31, and a single family member resides on chromosome 5p15(Shi, et al., Mol. Biol Evol 20:805-814, 2003). These genes all containa single coding exon (approximately 1 kb in length) that encodes areceptor averaging approximately 300 amino acids in length.

Individual members of the T2R family exhibit 30%-70% amino acididentity. The most highly conserved sequence motifs reside in the firstand last transmembrane segments, and also in the second cytoplasmicloop. The most divergent regions are the extracellular segments,extending partway into the transmembrane helices, possibly reflectingthe need to recognize structurally diverse ligands.

Taste sensitivity to the bitter compound phenylthiocarbamide (PTC) andrelated chemicals is bimodally distributed, and virtually all humanpopulations tested to date contain some people who can (tasters) andsome people who cannot taste (nontasters) PTC. The frequency of tastersin North Americans of European ancestry is about 70%. The PTC tastereceptor encoded on chromosome 7 was recently identified as a tastereceptor that mediates the bitter taste of at least PTC (Kim et al.,Science 299:1221-1225, 2003).

Although PTC itself has not been found in nature, the ability to tastePTC is correlated strongly with the ability to taste other naturallyoccurring bitter substances, many of which are toxic (Harris and Kalmus,Ann Eugen 15:32-45, 1949; Barnicot et al., Ann Eugen 16:119-128, 19;Tepper, Am J Hum Genet 63:1271-1276, 1998). Furthermore, variation inPTC taste sensitivity has been correlated with dietary preferences thatmay have significant health effects (Bartoshuk et al. 1994). Forexample, PTC is similar in structure to isothiocyanates (compoundscontaining the group N—C═S) and goitrin, both of which are bittersubstances found in cruciferous vegetables like cabbage and broccoli(Tepper, Am J Hum Genet 63:1271-1276, 1998). Variable aversions to thesecompounds have been implicated in the variable rates ofthyroid-deficiency disease in PTC tasters and nontasters, withnontasters being more susceptible (Drewnowski and Rock, Am J Clin Nutr62:506-511, 1995).

Identifying receptor-ligand relationships for T2Rs has been difficult,and the nature of the ligand that binds to each receptor and initiatesbitter taste perception is known for only a few of these receptors. Inhumans, in vitro cell based assays have shown that T2R16 responds tosalicin and other beta-glucopyranosides and T2R10 displays activity uponexposure to strychnine (Bufe, et al., Nat. Genet. 32:397-401, 2002). Analternative human genetic approach has revealed that T2R38 PTC) encodesthe receptor for phenylthiocarbamide, a classic variant trait in humans(Kim, et al., Science 299:1221-1225, 2003). The bitter tastant ligandsthat activate the remaining 22 human T2R proteins are not wellcharacterized.

SUMMARY OF THE DISCLOSURE

This disclosure provides a comprehensive collection of single nucleotidepolymorphisms (SNPs) in bitter taste receptor (T2R) genes (FIG. 1). Itis believed that a portion of these SNPs define biologically relevantdifference between different alleles of the bitter taste receptor genes.Included in the disclosure are sub-sets of the bitter taste receptorSNPs that represent conserved, non-conserved, silent, and truncationmutations in the corresponding proteins, as well as individual allelicsequences for the various bitter taste receptor genes.

The disclosure further provides methods for using the correspondingallelic variants of the taste receptor genes, alone or in variouscombinations, to test a subject's bitter tasting profile, and toidentity and analyze compounds that interact with and/or influencebitter tastes in subjects.

Also provided is a substantially comprehensive set of haplotypes fornearly all of the T2R bitter taste receptors (T2R1, T2R3, T2R4, T2R5,T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40,T2R41, T2R43, T2R44, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60).Details of the haplotypes, and the T2R isoforms encoded thereby, areprovided in Table 7.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (including pages 1-5) is a table showing SNPs identified in theindicated T2R bitter taste receptor genes.

FIG. 2 is a graph showing the distribution of cSNPs among the fivepopulation samples. The cSNPs were categorized as to whether they werevariable in one, two, three, four, or all five populations. Populationcodes are CAM, Cameroonians; AME, Amerindians; JAP, Japanese; HUG,Hungarians; PYG; Pygmies.

FIG. 3 is a graph showing the distributions of Tajima's D statistic.Dotted line indicates theoretical expectation under the assumption thathuman population sizes have been constant. Dashed line indicatestheoretical expectation under the assumption that the human populationsizes increased from 10,000 to 1,000,000, 100,000 years ago. Observedfractions were calculated across all genes (EGP and T2R). EGP genes wereresequenced in the 90-member NIH polymorphism discovery resource as partof the Environmental Genome Project. These genes encode proteins thoughtto be important in mediating the interface between the human body andthe environment. Observed T2R genes are the genes resequenced for thisstudy.

FIG. 4 is a minimum spanning tree of T2R49 haplotypes. Each circlerepresents a haplotype, the area of the circle represents the haplotypefrequency, and shading indicates the fraction at which the haplotype wasobserved in each continental sample. Each slash represents onenucleotide substitution. Amino acid substitutions are denoted withletter-number combinations. Europe and Africa are dominated by Cluster Iwhile Asia and Amerindian are dominated by Cluster 2.

SEQUENCE LISTING

The DNA and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows the coding nucleic acid of the bitter taste receptorgene T2R1, and the protein encoded thereby. Two SNPs are indicated.

SEQ ID NO: 2 shows the protein sequence of the T2R1 bitter tastereceptor.

SEQ ID NO: 3 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R3, and the protein encoded thereby. Three SNPs areindicated.

SEQ ID NO: 4 shows the protein sequence of the T2R3 bitter tastereceptor.

SEQ ID NO: 5 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R4, and the protein encoded thereby. Six SNPs areindicated.

SEQ ID NO: 6 shows the protein sequence of the T2R4 bitter tastereceptor.

SEQ ID NO: 7 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R5, and the protein encoded thereby. Six SNPs areindicated.

SEQ ID NO: 8 shows the protein sequence of the T2R5 bitter tastereceptor.

SEQ ID NO: 9 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R7, and the protein encoded thereby. One SNP isindicated.

SEQ ID NO: 10 shows the protein sequence of the T2R7 bitter tastereceptor.

SEQ ID NO: 11 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R8, and the protein encoded thereby. Four SNPs areindicated.

SEQ ID NO: 12 shows the protein sequence of the T2R8 bitter tastereceptor.

SEQ ID NO: 13 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R9, and the protein encoded thereby. Five SNPs areindicated.

SEQ ID NO: 14 shows the protein sequence of the T2R9 bitter tastereceptor.

SEQ ID NO: 15 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R10, and the protein encoded thereby. Five SNPs areindicated.

SEQ ID NO: 16 shows the protein sequence of the T2R10 bitter tastereceptor.

SEQ ID NO: 17 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R13, and the protein encoded thereby. One SNP isindicated.

SEQ ID NO: 18 shows the protein sequence of the T2R13 bitter tastereceptor.

SEQ ID NO: 19 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R14, and the protein encoded thereby. Two SNPs areindicated.

SEQ ID NO: 20 shows the protein sequence of the T2R14 bitter tastereceptor.

SEQ ID NO: 21 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R16, and the protein encoded thereby. Seven SNPs areindicated.

SEQ ID NO: 22 shows the protein sequence of the T2R16 bitter tastereceptor.

SEQ ID NO: 23 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R38, and the protein encoded thereby. Five SNPs areindicated.

SEQ ID NO: 24 shows the protein sequence of the T2R38 bitter tastereceptor, also known as the PTC taste receptor.

SEQ ID NO: 25 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R39, and the protein encoded thereby. Two SNPs areindicated.

SEQ ID NO: 26 shows the protein sequence of the T2R39 bitter tastereceptor.

SEQ ID NO: 27 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R40, and the protein encoded thereby. Two SNPs areindicated.

SEQ ID NO: 28 shows the protein sequence of the T2R40 bitter tastereceptor.

SEQ ID NO: 29 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R41, and the protein encoded thereby. Three SNPs areindicated.

SEQ ID NO: 30 shows the protein sequence of the T2R41 bitter tastereceptor.

SEQ ID NO: 31 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R43 (GenBank Accession No. AF494237), and the proteinencoded thereby. Ten SNPs are indicated.

SEQ ID NO: 32 shows the protein sequence of the T2R43 bitter tastereceptor.

SEQ ID NO: 33 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R44, and the protein encoded thereby. Ten SNPs areindicated.

SEQ ID NO: 34 shows the protein sequence of the T2R44 bitter tastereceptor.

SEQ ID NO: 35 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R46, and the protein encoded thereby. Four SNPs areindicated.

SEQ ID NO: 36 shows the protein sequence of the T2R46 bitter tastereceptor.

SEQ ID NO: 37 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R47, and the protein encoded thereby.

SEQ ID NO: 38 shows the protein sequence of the T2R47 bitter tastereceptor.

SEQ ID NO: 39 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R48, and the protein encoded thereby. Ten SNPs areindicated.

SEQ ID NO: 40 shows the protein sequence of the T2R48 bitter tastereceptor.

SEQ ID NO: 41 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R49, and the protein encoded thereby. Ten SNPs areindicated.

SEQ ID NO: 42 shows the protein sequence of the T2R49 bitter tastereceptor.

SEQ ID NO: 43 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R50, and the protein encoded thereby.

SEQ ID NO: 44 shows the protein sequence of the T2R50 bitter tastereceptor.

SEQ ID NO: 45 shows the coding nucleic acid sequence of bitter tastereceptor gene T2R60, and the protein encoded thereby. Two SNPs areindicated.

SEQ ID NO: 46 shows the protein sequence of the T2R60 bitter tastereceptor.

SEQ ID NOs: 47 (GenBank Accession No. AF227129), 49, and 51 (GenBankAccession No. AC026787.5) show the coding nucleic acid sequence ofhaplotypes of the T2R1 bitter taste receptor gene, and the proteinsencoded thereby.

SEQ ID NOs: 48, 50, and 52 show the protein sequences of the haplotypesof the T2R1 bitter taste receptor.

SEQ ID NOs: 53 (GenBank Accession No. AF227130) and 55 show the codingnucleic acid sequence of haplotypes of the T2R3 bitter taste receptorgene, and the proteins encoded thereby.

SEQ ID NOs: 54 and 56 show the protein sequences of the haplotypes ofthe T2R3 bitter taste receptor.

SEQ ID NOs: 57, 59, 61 (GenBank Accession No. AF227131), 63, 65, 67, 69,and 71 show the coding nucleic acid sequence of haplotypes of the T2R4bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 58, 60, 62, 64, 66, 68, 70, and 72 show the proteinsequences of the haplotypes of the T2R4 bitter taste receptor.

SEQ ID) NOs: 73 (GenBank Accession No. AF227132), 75, 77, 79, 81, 83,and 85 show the coding nucleic acid sequence of haplotypes of the T2R5bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 74, 76, 78, 80, 82, 84, and 86 show the protein sequences ofthe haplotypes of the T2R5 bitter taste receptor.

SEQ ID NOs: 87 (GenBank Accession No. AF227133), 89, 91, 93, and 95 showthe coding nucleic acid sequence of haplotypes of the T2R7 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 88, 90, 92, 94, and 96 show the protein sequences of thehaplotypes of the T2R7 bitter taste receptor.

SEQ ID NOs: 97 (GenBank Accession No. AF227134), 99, 101, 103, 105, and107 show the coding nucleic acid sequence of haplotypes of the T2R8bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 98, 100, 102, 104, 106, and 108 show the protein sequencesof the haplotypes of the T2R8 bitter taste receptor.

SEQ ID NOs: 109 (GenBank Accession No. AF227135), 111, 113, 115, 117,119, 121, and 123 show the coding nucleic acid sequence of haplotypes ofthe T2R9 bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 110, 112, 114, 116, 118, 120, 122, and 124 show the proteinsequences of the haplotypes of the T2R9 bitter taste receptor.

SEQ ID NOs: 125, 127, 129, and 131 (GenBank Accession No. AF227136) showthe coding nucleic acid sequence of haplotypes of the T2R10 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 126, 128, 130, and 132 show the protein sequences of thehaplotypes of the T2R10 bitter taste receptor.

SEQ ID NOs: 133 (GenBank Accession No. AF227137) and 135 show the codingnucleic acid sequence of haplotypes of the T2R13 bitter taste receptorgene, and the proteins encoded thereby.

SEQ ID NOs: 134 and 136 show the protein sequences of the haplotypes ofthe T2R13 bitter taste receptor.

SEQ ID NOs: 137 (GenBank Accession No. AF227138), 139, and 141 show thecoding nucleic acid sequence of haplotypes of the T2R14 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 138, 140, and 142 show the protein sequences of thehaplotypes of the T2R14 bitter taste receptor.

SEQ ID NOs: 143 (GenBank Accession No. CQ740130.1), 145 (GenBankAccession No. AF227139), 147, 149, and 151 show the coding nucleic acidsequence of haplotypes of the T2R16 bitter taste receptor gene, and theproteins encoded thereby.

SEQ ID NOs: 144, 146, 148, 150, and 152 show the protein sequences ofthe haplotypes of the T2R16 bitter taste receptor.

SEQ ID NOs: 153 (GenBank Accession No. AY258597.1), 155, 157, 159(GenBank Accession Nos. AX647247.1 and AY114095.1), 161, 163, and 165(GenBank Accession No. AF494231) show the coding nucleic acid sequenceof haplotypes of the T2R38 bitter taste receptor gene, and the proteinsencoded thereby.

SEQ ID NOs: 154, 156, 158, 160, 162, 164, and 166 show the proteinsequences of the haplotypes of the T2R38 bitter taste receptor.

SEQ ID NOs: 167 (GenBank Accession No. AF494230) and 169 show the codingnucleic acid sequence of haplotypes of the T2R39 bitter taste receptorgene, and the proteins encoded thereby.

SEQ ID NOs: 168 and 170 show the protein sequences of the haplotypes ofthe T2R39 bitter taste receptor.

SEQ ID NOs: 171 (GenBank Accession No. AF494229), 173, 175, and 179 showthe coding nucleic acid sequence of haplotypes of the T2R40 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 172, 174, 176, and-180 show the protein sequences of thehaplotypes of the T2R40 bitter taste receptor.

SEQ ID NOs: 181, 183, and 185 (GenBank Accession No. AF494232) show thecoding nucleic acid sequence of haplotypes of the T2R41 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ED NOs: 182, 184, and 186 show the protein sequences of thehaplotypes of the T2R41 bitter taste receptor.

SEQ ID NOs: 187, 189, 191, 193 (GenBank Accession No. AF494228), 195(GenBank Accession No. AX647301.1 and AC018630.40), 197, and 199 showthe coding nucleic acid sequence of haplotypes of the T2R44 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 188, 190, 192, 194, 196, 198, and 200 show the proteinsequences of the haplotypes of the T2R44 bitter taste receptor.

SEQ ID NOs: 201, 203, 205, 207 (GenBank Accession No. AF494227), 209,and 211 show the coding nucleic acid sequence of haplotypes of the T2R46bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 202, 204, 206, 208, 210, and 212 show the protein sequencesof the haplotypes of the T2R46 bitter taste receptor.

SEQ ID NOs: 213, 215 (GenBank Accession No. AF494233), 217, and 219 showthe coding nucleic acid sequence of haplotypes of the T2R47 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 214, 216, 218, and 220 show the protein sequences of thehaplotypes of the T2R47 bitter taste receptor.

SEQ ID NOs: 221 (GenBank Accession no. CQ800016.1), 223 (GenBankAccession No. AF494234), 225, 227, 229, 231, 233, 235, and 237 show thecoding nucleic acid sequence of haplotypes of the T2R48 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 222, 224, 226, 228, 230, 232, 234, 236, and 238 show theprotein sequences of the haplotypes of the T2R48 bitter taste receptor.

SEQ ID NOs: 239 (GenBank Accession No. AF494236), 241, 243, 245, 247,249 and 251 show the coding nucleic acid sequence of haplotypes of theT2R49 bitter taste receptor gene, and the proteins encoded thereby.

SEQ ID NOs: 240, 242, 244, 246, 248, 250, and 252 show the proteinsequences of the haplotypes of the T2R49 bitter taste receptor.

SEQ ID NOs: 253, 255 (GenBank Accession No. AF494235), 257, and 259 showthe coding nucleic acid sequence of haplotypes of the T2R50 bitter tastereceptor gene, and the proteins encoded thereby.

SEQ ID NOs: 254, 256, 258 and 260 show the protein sequences of thehaplotypes of the T2R50 bitter taste receptor.

SEQ ID NOs: 261 (GenBank Accession No. AY114094) and 263 show the codingnucleic acid sequence of haplotypes of the T2R60 bitter taste receptorgene, and the proteins encoded thereby.

SEQ ID NOs: 262 and 264 show the protein sequences of the haplotypes ofthe T2R60 bitter taste receptor.

DETAILED DESCRIPTION Abbreviations 2D-PAGE two-dimensionalpolyacrylamide gel electrophoresis ASO allele-specific oligonucleotideASOH allele-specific oligonucleotide hybridization DASH dynamicallele-specific hybridization ELISA enzyme-linked immunosorbant assayHPLC high pressure liquid chromatography MALDI-TOF matrix-assisted laserdesorption/ionization time-of-flight PCR polymerase chain reactionRT-PCR reverse-transcription polymerase chain reaction SNP singlenucleotide polymorphism SSCP single-strand conformation polymorphismII. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Addressable: Capable of being reliably and consistently located andidentified, as in an addressable location on an array.

Allele: A particular form of a genetic locus, distinguished from otherforms by its specific nucleotide sequence.

Amplified RNA (amRNA): A molecule of RNA generated through in vitrotranscription with T7 or other promoter region attached to the 5′ end ofthe template.

Antisense, Sense, and Antigene: Double-stranded DNA (dsDNA) has twostrands, a 5′->3′ strand, referred to as the plus strand, and a 3′->5′strand (the reverse complement), referred to as the minus strand.Because RNA polymerase adds nucleic acids in a 5′->3′ direction, theminus strand of the DNA serves as the template for the RNA duringtranscription. Thus, the RNA formed will have a sequence complementaryto the minus strand and identical to the plus strand (except that U issubstituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a dsDNAtarget.

Array: An arrangement of molecules, particularly biologicalmacromolecules (such as polypeptides or nucleic acids) or biologicalsamples (such as tissue sections) in addressable locations on asubstrate, usually a flat substrate such as a membrane, plate or slide.The array may be regular (arranged in uniform rows and columns, forinstance) or irregular. The number of addressable locations on the arraycan vary, for example from a few (such as three) to more than 50, 100,200, 500, 1000, 10,000, or more. A “microarray” is an array that isminiaturized to such an extent that it benefits from microscopicexamination for evaluation.

Within an array, each arrayed molecule (e.g., oligonucleotide) or sample(more generally, a “feature” of the array) is addressable, in that itslocation can be reliably and consistently determined within the at leasttwo dimensions on the array surface. Thus, in ordered arrays thelocation of each feature is usually assigned to a sample at the timewhen it is spotted onto or otherwise applied to the array surface, and akey may be provided in order to correlate each location with theappropriate feature.

Often, ordered arrays are arranged in a symmetrical grid pattern, butsamples could be arranged in other patterns (e.g., in radiallydistributed lines, spiral lines, or ordered clusters). Arrays arecomputer readable, in that a computer can be programmed to correlate aparticular address on the array with information (such as identificationof the arrayed sample and hybridization or binding data, including forinstance signal intensity). In some examples of computer readable arrayformats, the individual spots on the array surface will be arrangedregularly, for instance in a Cartesian grid pattern, that can becorrelated to address information by a computer.

The sample application spot (or feature) on an array may assume manydifferent shapes. Thus, though the term “spot” is used herein, it refersgenerally to a localized deposit of nucleic acid or other biomolecule,and is not limited to a round or substantially round region. Forinstance, substantially square regions of application can be used witharrays, as can be regions that are substantially rectangular (such as aslot blot-type application), or triangular, oval, irregular, and soforth. The shape of the array substrate itself is also immaterial,though it is usually substantially flat and may be rectangular or squarein general shape.

Binding or interaction: An association between two substances ormolecules, such as the hybridization of one nucleic acid molecule toanother (or itself). Disclosed arrays are used to detect binding of, insome embodiments, a labeled nucleic acid molecule (target) to animmobilized nucleic acid molecule (probe) in one or more features of thearray. A labeled target molecule “binds” to a nucleic acid molecule in aspot on an array if, after incubation of the (labeled) target molecule(usually in-solution or suspension) with or on the array for a period oftime (usually 5 minutes or more, for instance 10 minutes, 20 minutes, 30minutes, 60 minutes, 90 minutes, 120 minutes or more, for instance overnight or even 24 hours), a detectable amount of that molecule associateswith a nucleic acid feature of the array to such an extent that it isnot removed by being washed with a relatively low stringency buffer(e.g., higher salt (such as 3×SSC or higher), room temperature washes).Washing can be carried out, for instance, at room temperature, but othertemperatures (either higher or lower) also can be used. Targets willbind probe nucleic acid molecules within different features on the arrayto different extents, based at least on sequence homology, and the term“bind” encompasses both relatively weak and relatively stronginteractions. Thus, some binding will persist after the array is washedin a more stringent buffer (e.g., lower salt (such as about 0.5 to about1.5×SSC), 55-65° C. washes).

Where the probe and target molecules are both nucleic acids, binding ofthe test or reference molecule to a feature on the array can bediscussed in terms of the specific complementarity between the probe andthe target nucleic acids. Also contemplated herein are protein-basedarrays, where the probe molecules are or comprise proteins, and/or wherethe target molecules are or comprise proteins.

cDNA: A DNA molecule lacking internal, non-coding segments (e.g.,introns) and regulatory sequences that determine transcription. By wayof example, cDNA may be synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

DNA (deoxyribonucleic acid): DNA is a long chain polymer that containsthe genetic material of most living organisms (the genes of some virusesare made of ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which includes one of the fourbases (adenine, guanine, cytosine and thymine) bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term “codon” is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Enriched: The term “enriched” means that the concentration of a materialis at least about 2, 5, 10, 100, or 1000 times its natural concentration(for example), advantageously at least 0.01% by weight. Enrichedpreparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are alsocontemplated.

EST (Expressed Sequence Tag): A partial DNA or cDNA sequence, typicallyof between 200 and 2000 sequential nucleotides, obtained from a genomicor cDNA library, prepared from a selected cell, cell type, tissue ortissue type, organ or organism, which corresponds to an mRNA of a genefound in that library. An EST is generally a DNA molecule sequenced fromand shorter than the cDNA from which it is obtained.

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forexample Cy3, FITC, and Oregon Green, are characterized by their emissionat wavelengths generally in the range of 515-540λ. Red fluorophores, forexample Texas Red, Cy5 and tetramethylrhodamine, are characterized bytheir emission at wavelengths generally in the range of 590-690λ.

Examples of fluorophores are provided in U.S. Pat. No. 5,866,366 toNazarenko et al., and include for instance:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphtha ide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

Other contemplated fluorophores include GFP (green fluorescent protein),Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivativesthereof. Other fluorophores known to those skilled in the art may alsobe used.

Examples of fluorophores that are sensitive to ion concentration (suchas Ca²⁺ concentration or flux) include, but are not limited to,bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3) (B-438),Quin-2 (AM Q-1288), Fura-2 (AM F-1225), Indo-1 (AM I-1226), Fura-3 (AMF-1228), Fluo-3 (AM F-1241), Rhod-2, (AM R-1244), BAPTA (AM B-1205),5,5′-dimethyl BAPTA (AM D-1207), 4,4′-difluoro BAPTA (AM D-1216),5,5′-difluoro BAPTA (AM D-1209), 5,5′-dibromo BAPTA (AM D-1213), CalciumGreen (C-3011), Calcium Orange (C-3014), Calcium Crimson (C-3017),Fura-5 (F-3023), Fura-Red (F-3020), SBFI (S-1262), PBFI (P-1265),Mag-Fura-2 (AM M-1291), Mag-Indo-1 (AM M-1294), Mag-Quin-2 (AM M-1299),Mag-Quin-1 (AM M-1297), SPQ (M-440), SPA (S-460), Calcien(Fluorescein-bis(methyliminodiacetic acid); Fluorexon), and Quin-2(2-{[2-Bis-(carboxymethyl)amino-5-methylphenoxy]-methyl}-6-methoxy-8-bis-(carboxymethyl)aminoquinolinetetrapotassium salt). Many of these (and other calcium sensing compoundsknown to those of ordinary skill) are available, for instance, fromMolecular Probes, Invitrogen Detection Technologies, Eugene, Oreg.

Haplotype: The ordered, linear combination of polymorphisms (e.g., SNPs)in the sequence of each form of a gene (on individual chromosomes) thatexists in the population.

Haplotyping: Any process for determining one or more haplotypes in anindividual. Example methods are described herein, and may include use offamily pedigrees, molecular biological techniques, statisticalinference, or any combination thereof.

High throughput genomics: Application of genomic or genetic data oranalysis techniques that use microarrays or other genomic technologiesto rapidly identify large numbers of genes or proteins, or distinguishtheir structure, expression or function from normal or abnormal cells ortissues, or from cells or tissues of subjects with known or unknownphenotype and/or genotype.

Human Cells: Cells obtained from a member of the species Homo sapiens.The cells can be obtained from any source, for example peripheral blood,urine, saliva, tissue biopsy, surgical specimen, amniocentesis samplesand autopsy material. From these cells, genomic DNA, mRNA, cDNA, RNA,and/or protein can be isolated.

Hybridization: Nucleic acid molecules that are complementary to eachother hybridize by hydrogen bonding, which includes Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleotide units. For example, adenine and thymine are complementarynucleobases that pair through formation of hydrogen bonds.“Complementary” refers to sequence complementarity between twonucleotide units. For example, if a nucleotide unit at a certainposition of an oligonucleotide is capable of hydrogen bonding with anucleotide unit at the same position of a DNA or RNA molecule, then theoligonucleotides are complementary to each other at that position. Theoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotide units which can hydrogen bond with each other.

“Specifically hybridizable” and “complementary” are terms that indicatea sufficient degree of complementarity such that stable and specificbinding occurs between the oligonucleotide and the DNA or RNA or PNAtarget. An oligonucleotide need not be 100% complementary to its targetnucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, for example under physiological conditionsin the case of in vivo assays, or under conditions in which the assaysare performed.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na⁺ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989), chapters9 and 11, herein incorporated by reference.

Its vitro amplification: Techniques that increase the number of copiesof a nucleic acid molecule in a sample or specimen. An example of invitro amplification is the polymerase chain reaction, in which abiological sample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid.

The product of in vitro amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques.

Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Isoform: As used herein, the term isoform refers to a protein with aunique amino acid sequence specified by one haplotype of a gene, such asa T2R bitter receptor gene. By way of example, specific examples of T2Risoforms are shown in the sequence listing, SEQ ID NOs: 48-264 (even).

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Label: Detectable marker or reporter molecules, which can be attached tonucleic acids. Typical labels include fluorophores, radioactiveisotopes, ligands, chemiluminescent agents, metal sols and colloids, andenzymes. Methods for labeling and guidance in the choice of labelsuseful for various purposes are discussed, e.g., in Sambrook et al., inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., in Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley-Intersciences (1987).

Mutation: Any change of the DNA sequence within a gene or chromosome. Insome instances, a mutation will alter a characteristic or trait(phenotype), but this is not always the case. Types of mutations includebase substitution point mutations (e.g., transitions or transversions),deletions, and insertions. Missense mutations are those that introduce adifferent amino acid into the sequence of the encoded protein; nonsensemutations are those that introduce a new stop codon. In the case ofinsertions or deletions, mutations can be in-frame (not changing theframe of the overall sequence) or frame shift mutations, which mayresult in the misreading of a large number of codons (and often leads toabnormal termination of the encoded product due to the presence of astop codon in the alternative frame).

This term specifically encompasses variations that arise through somaticmutation, for instance those that are found only in disease cells, butnot constitutionally, in a given individual. Examples of suchsomatically-acquired variations include the point mutations thatfrequently result in altered function of various genes that are involvedin development of cancers. This term also encompasses DNA alterationsthat are present constitutionally, that alter the function of theencoded protein in a readily demonstrable manner, and that can beinherited by the children of an affected individual. In this respect,the term overlaps with “polymorphism,” as defined below, but generallyrefers to the subset of constitutional alterations.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompassing known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Nucleic acid array: An arrangement of nucleic acids (such as DNA or RNA)in assigned locations on a matrix, such as that found in cDNA arrays, oroligonucleotide arrays.

Nucleic acid molecules representing genes: Any nucleic acid, for exampleDNA (intron or exon or both), cDNA or RNA, of any length suitable foruse as a probe or other indicator molecule, and that is informativeabout the corresponding gene.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: A linear single-stranded polynucleotide sequenceranging in length from 2 to about 5,000 bases, for example apolynucleotide (such as DNA or RNA) which is at least 6 nucleotides, forexample at least 10, 12, 15, 18, 20, 25, 50, 100, 200, 1,000, or even5,000 nucleotides long. Oligonucleotides are often synthetic but canalso be produced from naturally occurring polynucleotides.

An oligonucleotide analog refers to moieties that function similarly tooligonucleotides but have non-naturally occurring portions. For example,oligonucleotide analogs can contain non-naturally occurring portions,such as altered sugar moieties or inter-sugar linkages, such as aphosphorothioate oligodeoxynucleotide. Functional analogs of naturallyoccurring polynucleotides can bind to RNA or DNA, and include peptidenucleic acid (PNA) molecules. Such analog molecules may also bind to orinteract with polypeptides or proteins.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Peptide Nucleic Acid (PNA): An oligonucleotide analog with a backbonecomprised of monomers coupled by amide (peptide) bonds, such as aminoacid monomers joined by peptide bonds.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with compositions provided herein are conventional. Byway of example, Martin, in Remington's Pharmaceutical Sciences,published by Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the nucleotides and proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polymorphism: Variant in a sequence of a gene, usually carried from onegeneration to another in a population. Polymorphisms can be thosevariations (nucleotide sequence differences) that, while having adifferent nucleotide sequence, produce functionally equivalent geneproducts, such as those variations generally found between individuals,different ethnic groups, geographic locations. The term polymorphismalso encompasses variations that produce gene products with alteredfunction, i.e., variants in the gene sequence that lead to gene productsthat are not functionally equivalent. This term also encompassesvariations that produce no gene product, an inactive gene product, orincreased or increased activity gene product.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule or protein that islinked to the variation (e.g., an alteration of a secondary structuresuch as a stem-loop, or an alteration of the binding affinity of thenucleic acid for associated molecules, such as polymerases, RNases, andso forth).

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on the nucleic acid molecules provided as indicators oftaste reception or likely taste reception. It is also appropriate togenerate probes and primers based on fragments or portions of thesenucleic acid molecules, particularly in order to distinguish between andamong different alleles and haplotypes within a single gene. Alsoappropriate are probes and primers specific for the reverse complementof these sequences, as well as probes and primers to 5′ or 3′ regions.

A probe comprises an isolated nucleic acid attached to a detectablelabel or other reporter molecule. Typical labels include radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescent orfluorescent agents, haptens, and enzymes. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed, e.g., in Sambrook et al. (In Molecular Cloning: A LaboratoryManual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocolsin Molecular Biology, John Wiley & Sons, New York, 1998).

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length. Longer DNAoligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or morein length. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then the primer extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other in vitro nucleic-acid amplification methodsknown in the art.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,1998), and Innis et al. (PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc., San Diego, Calif., 1990).Amplification primer pairs (for instance, for use with polymerase chainreaction amplification) can be derived from a known sequence such as anyof the bitter taste receptor sequences and specific alleles thereofdescribed herein, for example, by using computer programs intended forthat purpose such as PRIMER (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.).

One of ordinary skill in the art will appreciate that the specificity ofa particular probe or primer increases with its length. Thus, forexample, a primer comprising 30 consecutive nucleotides of a bittertaste receptor protein encoding nucleotide will anneal to a targetsequence, such as homolog of a designated taste receptor protein, with ahigher specificity than a corresponding primer of only 15 nucleotides.Thus, in order to obtain greater specificity, probes and primers can beselected that comprise at least 20, 23, 25, 30, 35, 40, 45, 50 or moreconsecutive nucleotides of a taste receptor gene.

Also provided are isolated nucleic acid molecules that comprisespecified lengths of bitter taste receptor-encoding nucleotidesequences. Such molecules may comprise at least 10, 15, 20, 23, 25, 30,35, 40, 45 or 50 or more (e.g., at least 100, 150, 200, 250, 300 and soforth) consecutive nucleotides of these sequences or more. Thesemolecules may be obtained from any region of the disclosed sequences(e.g., a specified nucleic acid may be apportioned into halves orquarters based on sequence length, and isolated nucleic acid moleculesmay be derived from the first or second, halves of the molecules, or anyof the four quarters, etc.). A cDNA or other encoding sequence also canbe divided into smaller regions, e.g. about eighths, sixteenths,twentieths, fiftieths, and so forth, with similar effect.

Another mode of division, provided by way of example, is to divide abitter taste receptor sequence based on the regions of the sequence thatare relatively more or less homologous to other bitter taste receptorsequences.

Nucleic acid molecules may be selected that comprise at least 10, 15,20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300 or more consecutivenucleotides of any of these or other portions of a bitter taste receptornucleic acid molecule or a specific allele thereof, such as thosedisclosed herein. Thus, representative nucleic acid molecules mightcomprise at least 10 consecutive nucleotides of the bitter tastereceptor nucleic acid coding sequence shown in any one of SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 39, 41,or 45 (indicating variable SNP positions), or SEQ ID NOs: 47-233 (oddnumbered sequences). More particularly, probes and primers in someembodiments are selected so that they overlap or reside adjacent to atleast one of the indicated SNPs indicated in the Sequence Listing or inFIG. 1 or Table 7

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified nucleicacid preparation is one in which the specified protein is more enrichedthan the nucleic acid is in its generative environment, for instancewithin a cell or in a biochemical reaction chamber. A preparation ofsubstantially pure nucleic acid may be purified such that the desirednucleic acid represents at least 50% of the total nucleic acid contentof the preparation. In certain embodiments, a substantially pure nucleicacid will represent at least 60%, at least 70%, at least 80%, at least85%, at least 90%, or at least 95% or more of the total nucleic acidcontent of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g. by genetic engineering techniques.

RNA: A typically linear polymer of ribonucleic acid monomers, linked byphosphodiester bonds. Naturally occurring RNA molecules fall into threeclasses, messenger (mRNA, which encodes proteins), ribosomal (rRNA,components of ribosomes), and transfer (tRNA, molecules responsible fortransferring amino acid monomers to the ribosome during proteinsynthesis). Total RNA refers to a heterogeneous mixture of all threetypes of RNA molecules.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of nucleic acid or aminoacid sequences will possess a relatively high degree of sequenceidentity when aligned using standard methods. This homology will be moresignificant when the orthologous proteins or nucleic acids are derivedfrom species which are more closely related (e.g., human and chimpanzeesequences), compared to species more distantly related (e.g., human andC. elegans sequences). Typically, orthologs are at least 50% identicalat the nucleotide level and at least 50% identical at the amino acidlevel when comparing human orthologous sequences.

Methods of alignment of sequences for comparison are well known. Variousprograms and alignment algorithms are described in: Smith & Waterman,Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol 48:443,1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al.Computer, Appls. Biosci. 8, 155-65, 1992; and Pearson et al., Meth. Mol.Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990,presents a detailed consideration of sequence alignment methods andhomology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastk, tblastn and tblastx.Each of these sources also provides a description of how to determinesequence identity using this program.

Homologous sequences are typically characterized by possession of atleast 60%, 70%, 75%, 80%, 90%, 95% or at least 98% sequence identitycounted over the full length alignment with a sequence using the NCBIBlast 2.0, gapped blastp set to default parameters. Queries searchedwith the blastn program are filtered with DUST (Hancock and Armstrong,Comput. Appl. Biosci. 10:67-70, 1994). It will be appreciated that thesesequence identity ranges are provided for guidance only; it is entirelypossible that strongly significant homologs could be obtained that falloutside of the ranges provided.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions, as described under “specific hybridization.”

Single Nucleotide Polymorphism (SNP): A single base (nucleotide)difference in a specific location in the DNA sequence among individualsin a population. A subset of SNPs give rise to changes in the encodedamino acid sequence; these are referred to as coding SNPs, or cSNPs.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the specified protein. By way of example, as usedherein, the term “X-protein specific binding agent” includes anti-Xprotein antibodies (and functional fragments thereof) and other agents(such as soluble receptors) that bind substantially only to the Xprotein (where “X” is a specified protein, or in some embodiments aspecified domain or form of a protein, such as a particular allelic formof a protein).

Anti-X protein antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988). The determination that aparticular agent binds substantially only to the specified protein mayreadily be made by using or adapting routine procedures. One suitable invitro assay makes use of the Western blotting procedure (described inmany standard texts, including Harlow and Lane (Antibodies, A LaboratoryManual, CSHL, New York, 1988)). Western blotting may be used todetermine that a given protein binding agent, such as an anti-X proteinmonoclonal antibody, binds substantially only to the X protein.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto a specified protein would be specific binding agents. These antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

Specific hybridization: Specific hybridization refers to the binding,duplexing, or hybridizing of a molecule only or substantially only to aparticular nucleotide sequence when that sequence is present in acomplex mixture (e.g. total cellular DNA or RNA). Specific hybridizationmay also occur under conditions of varying stringency.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing DNA used.Generally, the temperature of hybridization and the ionic strength(especially the Na+ concentration) of the hybridization buffer willdetermine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al (In: Molecular Cloning. ALaboratory Manual, Cold Spring Harbor, New York, 1989 ch. 9 and 11). Byway of illustration only, a hybridization experiment may be performed byhybridization of a DNA molecule to a target DNA molecule which has beenelectrophoresed in an agarose gel and transferred to a nitrocellulosemembrane by Southern blotting (Southern, J. Mol. Biol. 98:503, 1975), atechnique well known in the art and described in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York,1989).

Traditional hybridization with a target nucleic acid molecule labeledwith [³²P]-dCTP is generally carried out in a solution of high ionicstrength such as 6×SSC at a temperature that is 20-25° C. below themelting temperature, T_(m), described below. For Southern hybridizationexperiments where the target DNA molecule on the Southern blot contains10 ng of DNA or more, hybridization is typically carried out for 6-8hours using 1-2 ng/ml radiolabeled probe (of specific activity equal to10⁹ CPM/μg or greater). Following hybridization, the nitrocellulosefilter is washed to remove background hybridization. The washingconditions should be as stringent as possible to remove backgroundhybridization but to retain a specific hybridization signal.

The term T_(m) represents the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target sequence hybridize to the target sequence atequilibrium. Because the target sequences are generally present inexcess, at T_(m) 50% of the probes are occupied at equilibrium. TheT_(m) of such a hybrid molecule may be estimated from the followingequation (Bolton and McCarthy, Proc. Natl. Acad. Sci. USA 48:1390,1962):T _(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−0.63(%formamide)−(600/l)where l=the length of the hybrid in base pairs.

This equation is valid for concentrations of Na⁺ in the range of 0.01 Mto 0.4 M, and it is less accurate for calculations of Tm in solutions ofhigher [Na⁺]. The equation is also primarily valid for DNAs whose G+Ccontent is in the range of 30% to 75%, and it applies to hybrids greaterthan 100 nucleotides in length (the behavior of oligonucleotide probesis described in detail in Ch. 11 of Sambrook et al. (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Thus, by way of example, for a 150 base pair DNA probe derived from acDNA (with a hypothetical % GC of 45%), a calculation of hybridizationconditions required to give particular stringencies may be made asfollows: For this example, it is assumed that the filter will be washedin 0.3×SSC solution following hybridization, thereby: [Na+]=0.045 M; %GC=45%; Formamide concentration=0; 1=150 base pairs;Tm=81.5−16.6(log₁₀[Na+])+(0.41×45)−(600/150); and so Tm=74.4° C.

The T_(m) of double-stranded DNA decreases by 1-1.5° C. with every 1%decrease in homology (Bonner et al., J. Mol. Biol. 81:123, 1973).Therefore, for this given example, washing the filter in 0.3×SSC at59.4-64.4° C. will produce a stringency of hybridization equivalent to90%; that is, DNA molecules with more than 10% sequence variationrelative to the target cDNA will not hybridize. Alternatively, washingthe hybridized filter in 0.3×SSC at a temperature of 65.4-68.4° C. willyield a hybridization stringency of 94%; that is, DNA molecules withmore than 6% sequence variation relative to the target cDNA moleculewill not hybridize. The above example is given entirely by way oftheoretical illustration. It will be appreciated that otherhybridization techniques may be utilized and that variations inexperimental conditions will necessitate alternative calculations forstringency.

Stringent conditions may be defined as those under which DNA moleculeswith more than 25%, 15%, 10%, 6% or 2% sequence variation (also termed“mismatch”) will not hybridize. Stringent conditions are sequencedependent and are different in different circumstances. Longer sequenceshybridize specifically at higher temperatures. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH. An example of stringent conditions is a salt concentration of atleast about 0.01 to 1.0 M Na ion concentration (or other salts) at pH7.0 to 8.3 and a temperature of at least about 30° C. for short probes(e.g. 10 to 50 nucleotides). Stringent conditions can also be achievedwith the addition of destabilizing agents such as formamide. Forexample, conditions of 5×SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mMEDTA, pH 7.4) and a temperature of 25-30° C. are suitable forallele-specific probe hybridizations.

A perfectly matched probe has a sequence perfectly complementary to aparticular target sequence. The test probe is typically perfectlycomplementary to a portion (subsequence) of the target sequence. Theterm “mismatch probe” refers to probes whose sequence is deliberatelyselected not to be perfectly complementary to a particular targetsequence.

Transcription levels can be quantitated absolutely or relatively.Absolute quantitation can be accomplished by inclusion of knownconcentrations of one or more target nucleic acids (for example controlnucleic acids or with a known amount the target nucleic acidsthemselves) and referencing the hybridization intensity of unknowns withthe known target nucleic acids (for example by generation of a standardcurve).

Subject: Living, multicellular vertebrate organisms, a category thatincludes both human and veterinary subjects for example, mammals, birdsand primates.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview, Variants of Human Taste Receptor Genes

The inventors herein have discovered many novel polymorphic sites(polymorphisms, SNPs) in the T2R genes. These SNPs are listed in FIG. 1.In addition, the inventors have determined the haplotypes for 22 of theT2R genes; these haplotypes are listed in Table 7. The haplotypes wereidentified from related individuals from the Utah Genetic ReferenceProject (consisting of individuals of Northern European ancestry),unrelated individuals from the NIH (of European, Asian, AfricanAmerican, and Native American ancestry), and unrelated individuals infive different geographic populations, including Cameroonians,Amerindians, Japanese, Hungarians, and Pygmies. Distributions andfrequency of SNPs and haplotypes in the various populations are shownbelow, for instance in Tables 2, 3, 4, and 5B (SNPs and haplotypes forT2R38), and Table 7B (haplotypes for 22 T2R genes). Each of theidentified T2R haplotypes defines a naturally occurring variant(isoforms) of the corresponding T2R gene that exists in a humanpopulation.

Thus, in one embodiment there are provided methods, compositions andkits for genotyping one or more T2R gene(s) in an individual. Thegenotyping method comprises identifying the nucleotide pair that ispresent at one or more variant sites selected from the group listed inFIG. 1, in both copies of the selected T2R gene(s) from the individual.Examples of such methods further comprise identifying the nucleotidepairs at all variant sites within any one T2R gene. Specificcontemplated genotyping compositions comprise an oligonucleotide probeor primer that overlaps and is designed to specifically hybridize to atarget region containing, or adjacent to, one of the listed T2R SNPsites, for instance specifically one of the SNPs that is referred to inFIG. 1 as newly identified by the inventors. A representative genotypingkit comprises one or more oligonucleotide(s) designed to genotype one ormore of the T2R SNP sites. Examples of such kits include at least oneoligonucleotide designed to genotype a single T2R gene at all identifiedSNP sites (which is also useful in haplotying the individual). Otherexamples of such kits include at least one oligonucleotide designed togenotype at least one SNP within each of the 23 provided T2R genes. Onespecific example is a kit that comprises at least one oligonucleotidedesigned to genotype each and every SNP described herein. The providedgenotyping methods, compositions, and kits are useful, for instance, foridentifying an individual, or collection of individuals, that has one ofthe genotypes or haplotypes described herein.

Also provided herein are methods for haplotyping 22 T2R genes, singly orin combination with two or more of the set, in an individual. Inexamples of such methods, the method comprises determining, the identityof the nucleotide at one or more SNP sites (such as those listed inFIG. 1) for one copy or both copies (also referred to as diplotyping) ofthe chosen T2R gene(s). In specific examples of such methods, it isdetermined whether at least one copy of at least one T2R gene in theindividual's AGTR1 gene corresponds to one of the haplotypes shown inTable 7, below.

It is specifically contemplated that more than one T2R gene can behaplotyped (or genotyped) in the individual. By way of example, all ofthe T2R genes listed herein (T2R1, T2R3, T2R4, T2R, T2R7, T2R8, T2R9,T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40, T2R41, T2R43, T2R44,T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60) may be haplotyped(or genotyped at one or more SNP positions) in a single individual.Alternatively, any subset of T2R genes may be haplotyped/genotyped.

For example, the haplotyping method can be used to validate a specificT2R protein, or isoform (as defined by the provided haplotypes) as acandidate target for a ligand, such as a bitter tasting compound, or ablocker or other compound that interferes with or influences perceptionof bitter taste. Determining for a particular population the frequencyof one or more of the individual T2R haplotypes or haplotype pairsdescribed herein will facilitate a decision on whether to pursue it as atarget for influencing taste perception, for instance to alter medicine,food or drink preparations, in a way particularly suited to a givenpopulation.

If variable T2R activity or tastant binding is associated withperception of (or failure to perceive) a bitter tastant, then one ormore T2R haplotypes or haplotype pairs is expected to be found at ahigher frequency in taster (or non-taster) cohorts than in appropriatelygenetically matched control individuals. This is illustrated herein withthe T2R38 gene (also referred to as the PTC receptor). The practitioneror other individual, without a priori knowledge as to the phenotypiceffect of any specific T2R haplotype or haplotype pair, can apply theinformation derived from detecting T2R haplotypes in an individual todecide whether modulating activity of the chosen T2R would be expectedto be useful in influencing taste in an individual or a population.Various methods are provided herein for testing whether a compound orligand interacts with a specific T2R isoform/variant, including ex vivosystems and in vivo systems. Some of these systems measure perceivedtaste or changes thereto directly; others measure an upstream signal fortaste perception, such as for instance release of intracellular calciumbased on the activity of the T2R or another protein in the tasteperception pathway.

The provided T2R SNPs and haplotypes are also useful in screening forcompounds targeting a T2R (or family of T2R) protein to influence aphenotype associated with the T2R isoform, such as perception of a tastesuch as a bitter taste. For example, detecting which of the T2Rhaplotypes disclosed herein are present in individual members of atarget population with enables the practitioner or other individual toscreen for a compound(s) that displays the highest desired agonist orantagonist activity for each of the T2R isoforms present in the targetpopulation, or the most common isoforms present in the targetpopulation. Thus, without requiring any a priori knowledge of thephenotypic effect of any particular T2R haplotype, the providedhaplotyping methods provide the practitioner or other individual with atool to identify lead compounds that are more likely to show efficacy ininfluencing taste perception.

The method for haplotyping one or more T2R gene(s) in an individual isalso useful in the design of trials of candidate compounds forinfluencing perception of taste, particularly bitter taste, thatpredicted to be associated with T2R activity. For example, instead ofrandomly assigning subjects to the test or control group as is typicallydone now, determining which of the T2R haplotype(s) disclosed herein arepresent in individuals in the study enables one to select thedistribution of T2R haplotypes and/or sets or T2R haplotypes to test andcontrol groups, thereby controlling any possible bias in the resultsthat could be introduced by a larger frequency of any one T2R haplotypeor set of haplotypes that had a previously unknown association withresponse to the tastant or other ligand being studied. Thus, with theinformation provided herein, one can more confidently rely on theresults of the trial, without needing to first determine the specificphenotypic effect of any T2R haplotype or haplotype pair.

Another embodiment provides a method for identifying an associationbetween a trait and a T2R genotype, haplotype, or set of haplotypes forone or more of the T2R genes described herein. The method comprisescomparing the frequency of the T2R genotype, haplotype, or set ofhaplotypes in a population exhibiting the trait (e.g., taste recognitionof a compound, or activation of the target T2R isoform) with thefrequency of the T2R genotype or haplotype in a reference population. Ahigher frequency of the T2R genotype, haplotype, or set of haplotypes inthe population having the trait than in the reference populationindicates the trait is associated with the T2R genotype, haplotype, orset of haplotypes. In examples of such methods, the T2R SNP is selectedfrom a SNP indicated in FIG. 1 as being newly identified by theinventors, or the T2R haplotype is selected from the haplotypes shown inTable 7. Such methods have applicability, for instance, in developingdiagnostic tests for taste perception and development and identificationof compounds useful for influencing taste, particularly perception ofbitter taste, for instance in a specific target population.

Yet another embodiment is an isolated polynucleotide comprising anucleotide sequence which is a polymorphic variant (allele) of areference sequence for a T2R gene, or a fragment thereof, particularly afragment of 10 or more contiguous nucleotides that overlap a SNPidentified herein. The reference sequence for each T2R gene is indicatedby GenBank Accession number herein, for instance in the briefdescription of Sequence Listing. Polymorphisms in T2R genes areindicated in FIG. 1, and particularly relevant are those SNPs indicatedas new in that figure. Specific contemplated herein are isolated nucleicacid molecules that comprise a nucleotide sequence for a T2R allele,wherein the nucleotide sequence is selected from SEQ ID NO: 3, 7, 9, 11,13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 39, 41, 45, 49, 55, 57, 59,63, 65, 67, 69, 71, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95,99, 101, 103,105, 107, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 135, 139,141, 147, 149, 151, 155, 157, 161, 163, 169, 173, 175, 177, 179, 181,183, 187, 189, 191, 197, 199, 201, 203, 205, 209, 211, 213, 217, 219,225, 227, 229, 231, 233, 235, 237, 241, 243, 245, 247, 249, 251, 253,257, 259, or 263.

Other embodiments provide recombinant expression vectors comprising atleast one of the T2R allele variants operably linked to expressionregulatory elements, and recombinant host cells transformed ortransfected with such an expression vector. The recombinant vector andhost cell may be used, for instance, to express a T2R isoform forprotein structure analysis and compound binding studies, as discussedmore fully herein.

Also provided are T2R polypeptide isoforms, which comprise a polymorphicvariant of a reference amino acid sequence for a T2R protein. Thereference sequence for each T2R protein is indicated by GenBankAccession number herein, for instance in the brief description ofSequence Listing. Polymorphisms in T2R proteins are indicated in FIG. 1,and particularly relevant are those cSNPs indicated as new in thatfigure, which cause a change in the protein sequence and thereforeresult in a new T2R isoform. T2R variants are useful in studying theeffect of the variation on the biological activity of the T2R, as wellas on the binding affinity of candidate compounds (e.g., tastants)targeting T2R for influence perception of bitter taste.

Also provided are T2R sequence anthologies, which are collections of T2Ralleles or isoforms found in a selected population. The population maybe any group of at least two individuals, including but not limited to areference population, a target population, a geographic population(e.g., based on continent, country, region, and so forth), a familypopulation, a clinical population, and a sex-selected population. A T2Rsequence anthology may comprise individual T2R haplotype nucleic acidmolecules stored in separate containers such as tubes, separate wells ofa microtiter plate and the like. Individual allele nucleic acidmolecules or isoforms, or groups of such molecules, in a T2R sequenceanthology, may be stored in any convenient and stable form, includingbut not limited to in buffered solutions, as DNA precipitates,freeze-dried preparations and the like. A specific contemplated T2Rsequence anthology comprises the set of haplotypes (or the encodedisoforms) shown in Table 7A. Also contemplated are anthologies thatcomprise subsets of T2R sequences, such as for instance a set of all ofthe haplotypes (or encoded isoforms) for a single T2R gene, or a set ofat least one haplotype (or encoded isoform) for each T2R gene, and soforth.

Another embodiment provides specific binding agents, such as antibodies,that recognize and specifically bind to one of the variant T2R proteinsdescribed herein.

Yet another embodiment is a nonhuman transgenic animal, comprising atleast one polymorphic genomic T2R variant allele described herein, aswell as methods for producing such animals. The transgenic animals areuseful for studying expression of the T2R isoforms in vivo, forscreening and testing of compositions targeted against the T2R protein,and for analyzing the effectiveness of agents and compounds forinfluencing taste, for instance blocking bitter taste, in a biologicalsystem.

Yet another embodiment is a computer system for storing and displayingpolymorphism, and particularly haplotype, data determined for T2R genesas described herein. A typical computer system includes a computerprocessing unit, a display, and a database containing the data.Representative T2R polymorphism data includes T2R SNPs (such as thoselisted in FIG. 1), T2R genotypes, T2R haplotypes (such as those listedin Table 7) and population or other information about the individuals inone or more populations.

IV. Representative Uses of T2R SNPs and Haplotypes

Identifying receptor-ligand relationships has been difficult and thenature of the ligand that binds to each receptor and initiates bittertaste perception is known for only a few of these receptors. In humans,in vitro studies have shown that T2R16 responds to salicin and otherbeta-glucopyranosides and T2R10 to strychnine, while using analternative human genetic approach has revealed that T2R38 encodes thereceptor for phenylthiocarbamide (PTC), a classic variant trait inhumans. Distinct phenotypes have been clearly associated only withspecific haplotypes of the PTC receptor and there are now five SNPsdescribed corresponding to seven haplotypes, including taster,non-taster and intermediate alleles (see, e.g., PCT/US02/23172,published as WO 03/008627, which is incorporated herein by reference).The non-taster allele may encode an isoform that serves as a functionalreceptor for another as yet unidentified toxic bitter substance. T2R38(PTC) studies suggest that there may be substantial additionalcomplexity in the task of identifying specific ligands for each bittertaste receptor, as different alleles of each gene may encode receptorsthat recognize different ligands. However merely identifying inisolation the many different DNA variants (SNPs) in these genes is lessuseful as these variants could possibly exist in a huge number ofdifferent genetically-linked combinations (haplotypes) able to encode acorrespondingly huge number of bitter taste receptors expressed on thesurface of the tongue. (The number of different possible proteinsincreases as the number (N) of different cSNPs (SNPs able to give riseto changes in amino acid sequence) in the coding sequence of the gene by2^(N), where N is the number of different cSNPs in the coding sequenceof the gene.)

Thus, the identification of T2R haplotypes provided herein is importantbecause the individual sequence variants (SNPs) in isolation do notdetermine the receptor protein produced in a cell. For example, thethree variant sites in T2R38 are capable of producing eight differentprotein isoforms, depending on the combination of variant forms presentat each of the three sites (2³=eight potential haplotype sequences). Inreality, those three sites produce only five different haplotypes; threepossible haplotypes do not exist in any population worldwide insofar aswe can determine. This becomes particularly important when a genecontains many coding sequence variants, such as T2R49. There are elevendifferent coding SNP's in this gene, which could occur together in over2000 possible combinations, potentially producing over 2000 differentforms of the T2R49 receptor protein. In fact, as described herein, thereare only seven identified haplotypes, and thus only seven out of the2000 different receptor forms for this gene actually exist in humans. Inthe 22 T2R genes analyzed, we have identified a total of 109 differentprotein coding haplotypes, and thus 109 different isoforms of the T2Rproteins.

It is believed that different T2R haplotypes encode receptor isoformswith different chemical specificities for bitter tastants/ligands,analogous to the situation we have shown exists for T2R38. Efforts arecurrently ongoing worldwide to identify bitter tastants/ligands for eachreceptor. The results presented herein indicate that this cannot beviewed as an effort to decode ligands for only 22 or 23 different T2Rgenes. Instead the real nature of the problem is to decode the ligand(s)for each of at least 109 different haplotypes. Experiments to identifyligand(s) (or blockers) for all possible haplotypes (which number manythousands, counting all 23 T2R genes together) are not practicable withcurrent technologies, nor are they necessary if the vast majority ofpossible haplotypes/isoforms do not in fact occur in nature. Ourinformation reveals the subset of possible haplotype sequences that areactually present in humans, and are thus worthy of further study. Thiswill enable more rapid and efficient de-orphanizing of each of the tastereceptors, as we have done for T2R38.

Also reported herein are T2R haplotype frequencies in each of severalpopulations, including Europeans, East Asians, and Africans. Thisinformation can be used to design foods and beverages for differentworldwide markets, in two ways. First, in food and beverage research anddevelopment, population-specific haplotype distribution information willallow the selection of panels of taste sensors in a rational andefficient manner. This information will also be useful in either pure invitro systems, or in panels of human volunteer tasters, who can begenotyped or selected using these discoveries. Second, knowledge of thegenetic underpinnings in individual taste preferences in targetpopulations will provide powerful predictive information for food andbeverage palatability in different populations. Thuspopulation-specific, and indeed even region-specific, anthologies ordatabases are now able to be developed using this information; these canprovide T2R haplotype frequencies in regional or local populations.These resources can be used to improve both development and marketingdecisions in the flavorings, food, and beverages industries.

Also provided based on the discoveries described herein are methods anddevices for high throughput analysis of T2R genotype and/or phenotype inan individual or group of individuals. A specific example of such a highthroughput device is a DNA or protein microarray, which contains acollection of two or more T2R alleles or SNP-specific oligonucleotides(in the case of a DNA microarray) or isoform proteins or variantfragments thereof (in the case of a protein microarray). Examples ofsuch arrays of molecules include at least one molecule representing eachof the 109 haplotypes listed in Table 7A. Specific example arraysinclude at least two sequences selected from SEQ ID NOs: 3, 7, 9, 11,13, 15, 19, 21, 23, 25, 27, 29, 31, 33, 35, 39, 41, 45, 49, 55, 57, 59,63, 65, 67, 69, 71, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 99, 101,103, 105, 107, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 135,139, 141, 147, 149, 151, 155, 157, 161, 163, 169, 173, 175, 177, 179,181, 183, 187, 189, 191, 197, 199, 201, 203, 205, 209, 211, 213, 217,219, 225, 227, 229, 231, 233, 235, 237, 241, 243, 245, 247, 249, 251,253, 257, 259, and 263, or an oligonucleotide comprising at least 6 orat least 10 contiguous nucleotides selected from one of these sequencesand which oligonucleotide overlaps at least one SNP as listed in FIG. 1.Other specific example arrays include at least five such sequences, atleast 10, at least 20, at least 30, at least 50 or more, including forinstance all 81 of the following: SEQ ID NOs: 49, 55, 57, 59, 63, 65,67, 69, 71, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 99, 101, 103, 105,107, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 135, 139, 141,147, 149, 151, 155, 157, 161, 163, 169, 173, 175, 177, 179, 181, 183,187, 189, 191, 197, 199, 201, 203, 205, 209, 211, 213, 217, 219, 225,227, 229, 231, 233, 235, 237, 241, 243, 245, 247, 249,251, 253, 257,259, and 263, or an oligonucleotide fragment of each of these sequenceswhich oligonucleotide overlaps at least one SNP form the sequence.

By way of example, such arrays can be used in genotyping and haplotypingof individuals or groups of individuals. In certain embodiments, theresults from such genotyping/haplotyping is used to select a cohort ofindividuals of known genotype/haplotype for at least one T2R receptor(or a combination of two or more T2R receptors, or all T2R receptors).These individuals could then be trained (as necessary) and used inflavor panel evaluation. Because the population of taste evaluators areof known (or partially known) genotype as relates to T2R receptor(s), arelatively small panel of tasters can provide results that can beextrapolated out to a large (e.g. commercially relevant) population.Such large population is beneficially characterized by the frequency ofoccurrence of specific T2R isoforms/haplotypes, so that panels can bematched to the expected taste preference(s) of the population. Theteachings herein enable such methods of extrapolating the bitter tastereceptor haplotype from a small group to a large (commercially relevant)population, thus representing a savings in time and cost. Such anapproach could be used, for instance, for “deorphanising” T2R receptorsfor specific bitter tastes/tastants or combinations thereof, forevaluating likely population response to tastants or blockers, or tocharacterize or develop new tastant molecules or blockers. This wouldallow decisions about population-specific taste variation to aiddecisions about worldwide marketing of specific flavorings and food andbeverage products.

Also contemplated are in vitro biochemical functional assays of T2Rtaste receptor function. Such studies employ a variety of differentassays, which produce information about G protein activation uponbinding of tastant ligands to T2R receptors. One long term goal of suchstudies is the development of an “artificial tongue” that could be usedto perform taste tests without the intervention of living humans astaste sensors.

V. Ex Vivo Uses of T2R Bitter Receptor Haplotype-Specific Isoforms

These haplotypes can be used to make protein expression constructionsand generate 109 unique T2R bitter receptor proteins. These proteins canbe arranged in a battery or an array to create a group of sensors forbitter tastants ligands. Such an array could be employed in largeparallel high-throughput systems, that would allow the testing of theeffects of bitter tastant ligands on all forms of all receptors withoutthe intervention of human tasters.

These expressed isoform receptors can be used in ex vivo reporter assaysof several types. One type is exemplified in the publication of Adler etal. Cell 100:693 (2000) (incorporated herein by reference in itsentirety). The method employs calcium-sensitive dyes to assay therelease of calcium from intracellular stores in response to G proteinactivation by ligand binding to the expressed T2R receptor protein.Another contemplated method employs direct measurement of G proteinactivation by binding of a radioactive, nonhydrolyzable analog of GTP ina cell-free reconstituted system containing G proteins, T2R receptor,and ligand, as described by Sainz et al. Abstracts of the XXVI Meetingof the Association for Chemoreception Sciences, 211:55, 2004(incorporated herein by reference). Either of these systems can be used,for instance in an array-based format, to identify or develop ligandsthat interact with T2R isoforms or sets of isoforms, or with specificT2R genes, as well as to identify agents that influence the binding ofsuch ligands. For instance, agents that reduce (e.g., block) the bindingof a ligand to a specific T2R isoform (or set thereof), or that competewith the binding of a known ligand, can be identified by a reduction insignal in a calcium-sensitive dye system, or by the reduction in bindingof the radioactive GTP analog. Agents that increase or enhance thebinding of a ligand can be identified by increased signals in eithersystem.

VI. Overview of Several Specific Embodiments

Encompassed herein are isolated T2R variant-specific nucleic acidmolecules, each of which comprise at least about 10 contiguousnucleotides that span (that is, include) at least one SNP identified asnew in FIG. 1. Also provided are arrays, which comprising two or moresuch nucleic acid molecules. By way of example, such arrays can compriseat least one nucleic acid molecule comprising at least about 10contiguous nucleotides from T2R1, T2R3, T2R4, T2R5, T2R7, T2R8, T2R9,T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40, T2R41, T2R43, T3R44,T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60, and spanning atleast one SNP identified as new in FIG. 1. Other examples of the arraycomprise at least one oligonucleotide from each T2R haplotype/allelelisted in Table 7. Specific examples of the arrays are in the format ofmicroarrays.

Also provided are collections of two of more isolated T2Rvariant-specific nucleic acid molecule (in other words, specific for atleast one variant position in a T2R gene described herein), each nucleicacid molecule in the collection comprising at least about 10 contiguousnucleotides spanning at least one T2R SNP position listed in Table 7.Examples of such collections comprise at least one isolated T2Rvariant-specific nucleic acid molecule from T2R1, T2R3, T2R4, T2R5,T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40,T2R41, T2R43, T2R44, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60. Othercollections comprise at least one isolated T2R variant-specific nucleicacid molecule from every SNP listed in Table 7. Still other collectionscomprise at least one isolated T2R variant-specific nucleic acidmolecule from each of SEQ ID NO: 49, 55, 57, 59, 63, 65, 67, 69, 71, 75,77, 79, 81, 83, 85, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 135, 139, 141, 147, 149, 151,155, 157, 161, 163, 169, 173, 175, 177, 179, 181, 183, 187, 189, 191,197, 199, 201, 203, 205, 209, 211, 213, 217, 219, 225, 227, 229, 231,233, 235, 237, 241, 243, 245, 247, 249, 251, 253, 257, 259, and 263. Forinstance, in examples of such collections, the isolated T2Rvariant-specific nucleic acid molecules have a sequence as shown in SEQID NO: 49, 55, 57, 59, 63, 65, 67, 69, 71, 75, 77, 79, 81, 83, 85, 89,91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 135, 139, 141, 147, 149, 151, 155, 157, 161, 163, 169,173, 175, 177, 179, 181, 183, 187, 189, 191, 197, 199, 201, 203, 205,209, 211, 213, 217, 219, 225, 227, 229, 231, 233, 235, 237, 241, 243,245, 247, 249, 251, 253, 257, 259, or 263.

Optionally, in collections provided herein, each nucleic acid moleculeis stored in a separate container. For instance, the separate containersin some embodiments are wells of a microtiter plate or equivalentthereof In other embodiments, the nucleic acid molecules of thecollections are affixed to a solid surface in an array, such as amicroarray.

In one embodiment, the microarray collection comprises nucleic acidmolecules having the sequence as set for in SEQ ID NO: 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259,261, and 263.

Other specific contemplated collections contain isolated T2Rvariant-specific nucleic acid molecules taken from a single T2R gene.For instance, collections are contemplated wherein the moleculescomprise: (a) SEQ ID NOs: 47, 49, and 51; (b) SEQ ID NOs: 53 and 55; (c)SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, and 71; (d) SEQ ID NOs: 73, 75,77, 79, 81, 83, and 85; (e) SEQ ID NOs: 87, 89, 91, 93, and 95; (f) SEQID NOs: 97, 99, 101, 103, 105, and 107; (g) SEQ ID NOs: 109, 111, 113,115, 117, 119, 121, and 123; (h) SEQ ID NOs: 125, 127, 129, and 131; (i)SEQ ID NOs: 133 and 135; (j) SEQ ID NOs: 137, 139, and 141; (k) SEQ IDNOs: 143, 145, 147, 149, and 151; (l) SEQ ID NOs: 153, 155, 157, 159,161, 163, and 165; (m) SEQ ID NOs: 167 and 169; (n) SEQ ID NOs: 171,173, 175, and 179; (o) SEQ ID NOs: 181, 183, and 185; (p) SEQ ID NOs:187, 189, 191, 193, 195, 197, and 199; (q) SEQ ID NOs: 201, 203, 205,207, 209, and 211; (r) SEQ ID NOs: 213, 215, 217, and 219; (s) SEQ IDNOs: 221, 223, 225, 227, 229, 231, 233, 235, and 237; (t) SEQ ID NOs:239, 241, 243, 245, 247, 249 and 251; (u) SEQ ID NOs: 253, 255, 257, and259; (v) SEQ ID NOs: 261 and 263; or (w) a combination of two or more of(a) through (v).

Also provided are isolated T2R polypeptide isoform fragment, such aspolypeptide fragments encoded by an isolated T2R variant-specificnucleic acid molecule that comprises at least about 10 contiguousnucleotides that span (that is, include) at least one SNP identified asnew in FIG. 1.

Another embodiment is an isolated T2R isoform polypeptide fragmentcomprising an amino acid sequence comprising at least 10 contiguousamino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 50, 56, 58, 60, 64, 66, 68, 70,72, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 100, 102, 104, 106, 108,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 136, 140, 142, 148,150, 152, 156, 158, 162, 164, 170, 174, 176, 178, 180, 182, 184, 188,190, 192, 198, 200, 202, 204, 206, 210, 212, 214, 218, 220, 226, 228,230, 232, 234, 236, 238, 242, 244, 246, 248, 250, 252, 254, 258, 260, or264, which fragment includes at least one amino acid variation as setforth in FIG. 1 or Table 7.

Also provided are isolated T2R polypeptide isoforms, which comprise anamino acid sequence selected from SEQ ID NO: 50, 56, 58, 60, 64, 66, 68,70, 72, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 100, 102, 104, 106, 108,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 136, 140, 142, 148,150, 152, 156, 158, 162, 164, 170, 174, 176, 178, 180, 182, 184, 188,190, 192, 198, 200, 202, 204, 206, 210, 212, 214, 218, 220, 226, 228,230, 232, 234, 236, 238, 242, 244, 246, 248, 250, 252, 254, 258, 260, or264, and isolated nucleic acid molecules encoding such T2R polypeptideisoforms, vectors comprising one of the isolated nucleic acid molecules,and host cells comprising such vectors.

Yet further embodiments are isolated nucleic acid molecules comprising anucleotide sequence for a T2R allele, wherein the nucleotide sequence isselected from SEQ ID NO: 49, 55, 57, 59, 63, 65, 67, 69, 71, 75, 77, 79,81, 83, 85, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 135, 139, 141, 147, 149, 151, 155, 157,161, 163, 169, 173, 175, 177, 179, 181, 183, 187, 189, 191, 197, 199,201, 203, 205, 209, 211, 213, 217, 219, 225, 227, 229, 231, 233, 235,237, 241, 243, 245, 247, 249, 251, 253, 257, 259, or 263, vectorscomprising one of the isolated nucleic acid molecules, and host cellscomprising such vectors.

A method of screening compounds useful for modulating bitter taste isalso provided. Such methods comprise contacting a test compound with ahost cell (such as a eukaryotic cell, for instance a HEK293 cell) ormembrane thereof that expresses a T2R taste receptor isoform encoded byan isolated nucleic acid molecule described herein; and detecting achange in the expression of the nucleotide sequence or a change inactivity of the T2R taste receptor, or detecting binding of the compoundto the T2R taste receptor or detecting a change in the electricalactivity of the host cell or a change in intracellular or extracellularcAMP, cGMP, IP3, or Ca²⁺ of the host cell. In certain embodiments, thegene product of said nucleotide sequence is fused to a sequence thatfacilitates localization to the cell membrane, wherein that sequence isat least 20 consecutive N terminal amino acids of a rhodopsin protein.In examples of the screening methods, a change in intracellular Ca²⁺ isdetected by measuring a change in a calcium-sensitive dye dependentfluorescence in the cell. In a preferred embodiment, a change inintracellular Ca²⁺ is detected by measuring a change in Fura-2fluorescence in the cell.

Another example of such a screening method is a high throughput method,which method comprises: contacting in parallel a test compound with acollection of host cells or membranes thereof each of which expresses adifferent T2R taste receptor isoform encoded by an isolated nucleic acidmolecule comprising a nucleotide sequence for a T2R allele, wherein thenucleotide sequence is selected from SEQ ID NO: 49, 55, 57, 59, 63, 65,67, 69, 71, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 99, 101, 103, 105,107, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 135, 139, 141,147, 149, 151, 155, 157, 161, 163, 169, 173, 175, 177, 179, 181, 183,187, 189, 191, 197, 199, 201, 203, 205, 209, 211, 213, 217, 219, 225,227, 229, 231, 233, 235, 237, 241, 243, 245, 247, 249, 251, 253, 257,259, and 263; and detecting a change in the expression of at least oneof the nucleotide sequences or a change in activity of at least one ofthe T2R taste receptors, or detecting binding of the compound to atleast one of the T2R taste receptors or detecting a change in theelectrical activity of at least one of the host cells or a change inintracellular or extracellular cAMP, cGMP, IP3, or Ca²⁺ of at least oneof the host cells. Optionally, the collection of host cells or membranesthereof are in the form of an array.

Another provided method is an in vivo method of screening compoundsuseful for modulating bitter taste, comprising: contacting a testcompound to a T2R taste receptor isoform encoded by an isolated T2Rnucleic acid molecule described herein; and detecting a change in theactivity of the T2R taste receptor, or detecting binding of the compoundto the T2R taste receptor. Examples of this method are high throughputmethods, and comprise: contacting in parallel a test compound with acollection of different T2R taste receptor isoforms encoded by theisolated nucleic acid molecules; and detecting a change in the activityof at least one of the T2R taste receptors, or detecting binding of thecompound to at least one of the T2R taste receptors. Optionally, thecollection of different T2R taste receptor isoforms are in the form ofan array.

Another embodiment provides a specific binding agent capable ofdiscriminating between or among two or more T2R polypeptide isoforms, orisoform specific fragments thereof. Examples of such specific bindingagents are antibodies, such as for instance, monoclonal antibodies.

Yet another provided method is a method of determining a T2R genotype ofa subject (e.g., genotyping or haplotying a subject), comprising:obtaining a test sample of DNA containing a T2R sequence of the subject;and determining whether the subject has a polymorphism in the T2Rsequence, wherein the polymorphism is selected from the SNPs referred toas new in FIG. 1.

Also provided is a method of identifying a plurality of individuals whoare genetically heterogeneous in at least one T2R gene, comprising:determining a T2R genotype for a plurality of subjects using the methodof claim 39; and selecting group of the subjects who are geneticallyheterogeneous in at least one T2R gene. Optionally, the plurality ofindividuals are selected to represent the genetic profile of ageographically defined population, such as for instance the geneticprofile of Europeans, East Asians, or Africans.

Also provided herein are kits. A first kit is provided for determiningwhether or not a subject has a selected T2R genotype or haplotype,comprising: a container comprising at least one oligonucleotide specificfor a T2R sequence comprising at least one SNP referred to as new inFIG. 1; and instructions for using the kit, the instructions indicatingsteps for: performing a method to detect the presence of variant T2Rnucleic acid in the sample; and analyzing data generated by the method,wherein the instructions indicate that presence of the variant nucleicacid in the sample indicates that the individual has the selected T2Rgenotype or haplotype. Optionally, such kits in some embodiments willfurther comprise a container that comprises a detectableoligonucleotide.

Further, a kit is provided for determining whether or not a subject hasa selected T2R genotype or haplotype, the kit comprising a containercomprising a T2R isoform-specific antibody; a container comprising anegative control sample; and instructions for using the kit, theinstructions indicating steps for: performing a test assay to detect aquantity of T2R isoform protein in a test sample of tissue and/or bodilyfluid from the subject, performing a negative control assay to detect aquantity of T2R isoform protein in the negative control sample; andcomparing data generated by the test assay and negative control assay,wherein the instructions indicate that a quantity of T2R isoform proteinin the test sample more than the quantity of T2R isoform protein in thenegative control sample indicates that the subject has the selected T2Rgenotype of haplotype, and wherein the T2R isoform-specific antibody isspecific for a T2R isoform having a sequence selected from SEQ ID NOs:50, 56, 58, 60, 64, 66, 68, 70, 72, 76, 78, 80, 82, 84, 86, 90, 92, 94,96, 100, 102, 104, 106, 108, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 136, 140, 142, 148, 150, 152, 156, 158, 162, 164, 170, 174,176, 178, 180, 182, 184, 188, 190, 192, 198, 200, 202, 204, 206, 210,212, 214, 218, 220, 226, 228, 230, 232, 234, 236, 238, 242, 244, 246,248, 250, 252, 254, 258, 260, and 264. Optionally, examples of such kitsfurther comprising a container that comprises a detectable antibody thatbinds to the antibody specific for the T2R isoform protein.

Another provided method is a method of screening for a compound usefulin influencing T2R taste perception in a mammal, comprising determiningif a test compound binds to or interacts with the T2R polypeptideisoform described herein or an isolated T2R polypeptide isoform fragmentspecific for a T2R variant, and selecting a compound that so binds. Incertain examples of this method, binding of the compound inhibits a T2Rprotein biological activity. In other examples, the compound stimulatesa T2R protein biological activity.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.In particular, other methods known to those of ordinary skill in the artcan be substituted for specific methods described herein. By way ofexample, additional methods for studying bitter taste receptors andcompounds that interact therewith are described in PCT/US02/23172(published as WO 03/008627), herein incorporated by reference in itsentirety.

EXAMPLES Example 1 Characterization of SNPs in the T2R (TAS2R) BitterTaste Receptor Gene PTC

The ability to taste the substance phenylthiocarbamide (PTC) has beenwidely used for genetic and anthropological studies, but genetic studieshave produced conflicting results and demonstrated complex inheritancefor this trait. We have identified a small region on chromosome 7q thatshows strong linkage disequilibrium between SNP markers and PTC tastesensitivity in unrelated subjects. This region contains a single genethat encodes a member of the TAS2R bitter taste receptor family.

This example describes the identification and analysis of singlenucleotide polymorphisms and haplotypes in the PTC gene. We identifiedthree coding SNP's giving rise to five haplotypes in this geneworldwide. These haplotypes completely explain the bimodal distributionof PTC taste sensitivity, thus accounting for the inheritance of theclassically defined taste insensitivity, and 55-85% of the variance inPTC sensitivity. Distinct phenotypes were associated with specifichaplotypes, demonstrating the direct influence of this gene on PTC tastesensitivity, and that variant sites interact with each other within theencoded gene product.

Methods and Materials:

PTC phenotype determinations. Subjects began tasting a solution of 1micromolar PTC (solution #14) and proceeded in 2-fold increasingconcentration increments (solutions 13, 12, 11 . . . ) until a bittertaste was perceived. Subjects then performed a blinded sorting testcontaining 3 cups of PTC solution and 3 cups of water. Raw tastethreshold was the most dilute solution at which the subject couldcorrectly sort all 6 cups. We also included a quinine thresholdmeasurement according to Blakeslee & Salmon (Proc. Natl. Acad. Sci. USA21, 84, 1935) to identify and exclude individuals with general deficitsin bitter taste (aguesia). For dichotomous assignment of phenotype, weconsidered individuals unable to taste PTC before solution #6, i.e. atconcentrations less than 267 micromolar PTC, to be non-tasters. Althoughthe classic method includes corrections for age and sex, analysis of ourraw PTC taste threshold data indicated only a modest sex effect, withfemales more sensitive than males (p=0.00324, proportion of varianceexplained=5.1%). No effect of age on PTC scores was observed. As aresult, raw PTC threshold scores were used for all analyses.

Research subjects. The Utah C.E.P.H. families were enrolled inconjunction with the Utah Genetic Reference Project under University ofUtah IRB approved protocol #6090-96, and consisted of individuals ofNorthern European ancestry. Subjects in the NIH replication sample wereenrolled under NIH/NINDS IRB approved protocol #DC-01-230, and were ofEuropean, Asian, African American, and Native American ancestry. HumanDiversity Panel DNAs (sub-Saharan African, Asian, and Southwest NativeAmerican) and primate DNAs were obtained from the Coriell CellRepository, Camden, N.J. The Utah sample consists of 27 familiescomprising 269 individuals; both haplotype and phenotype information wasavailable for 180 of these individuals. The NIH replication sample ofconsisted of 85 unrelated individuals of known haplotype and phenotype;51 were European, 5 Pakistani, 23 East Asian, and 6 African-American.One African-American is not considered in the analysis due to a rareAAV/AAI diplotype. His raw PTC score is 7.

Bioinformatics analyses. Bioinformatics analysis was performed with theNCBI Human Genome databases (available on the Web atncbi.nlm.nih.gov/genome/guide/human) and the Celera Discovery System(available online at cds.celera.com/cd). Gene finding was performed withBLASTX (available on the Web at ncbi.nlm.nih.gov/BLAST) and GENESCAN andFGENES software (GeneMachine, DIR, NIH, available online atgenome.nhgri.nih.gov/genemachine/). SNPs were developed using the SNPdatabase available on the Web at ncbi.nlm.nih.gov/SNP/).

PTC gene haplotyping. Haplotypes within the PTC gene were determined byperforming genomic PCR to obtain a 1195 bp product containing all 3variant sites, using primers as follows: F=5′ GCTTTGTGAGGAATCAGAGTTGT3′, R=5′ GAACGTACATTTACCTTTCTGCACT 3′. The mass PCR product from eachindividual was cloned into TopoTA vector (Clonetech), and singlecolonies which contained a single amplified haplotype were picked andsequenced.

QTL linkage analysis. Quantitative trait linkage analysis was performedusing SOLAR, Almassy and Blangero, Am. J. Hum. Genet. 62, 1198, 1998.The effect of PTC haplotypes on the linkage results was determined byperforming two multipoint linkage analyses: one using the raw PTC scoresand another using adjusted PTC scores, both with sex as a covariate. Thefirst analysis excluded diplotypes as covariates, the second includedthem. For the latter, adjusted scores were obtained by subtracting offthe mean of each diplotype group from the scores of individuals withthat particular diplotype.

Haplotype effect analysis. The effect of the PTC haplotypes, as well asthe covariates sex and age, on raw PTC scores was estimatedsimultaneously in a multivariate analysis using the program SOLAR²⁶.SOLAR estimates the proportion of variance explained by a covariate(e.g., the PTC diplotype) in the presence of background polygenicvariance, in this case estimated from residual familial correlation inthe phenotype. The program also takes into account non-independence ofsib genotypes. The confirmation sample of unrelated individuals wasanalyzed using multiple linear regression with sex and age as covariatesas well as Analysis of Variance.

GenBank Human candidate taste receptor gene TAS2R38 (GenBank accessionnumber AF494231) is identical to the sequence of the non-taster AVI formof the PTC gene, with the exception of nucleotide 557, which is an A(encoding Asn¹⁸⁶) in TAS2R38 and a T (encoding Ile¹⁸⁶) in PTC.

Material in this example was published as Kim et al., Science299:1221-1225, Feb. 21, 2003, which publication is incorporated hereinby reference in its entirety, including the supplemental materialpublished on-line at scienemag.org/cgi/content/full/299/5610/121/DC1.

Results and Discussion

The inability to taste PTC (Science 73:4, 1931; Guo and Reed, Ann. Hum.Biol. 28:111, 2001) was long believed to be a simple Mendelian recessivetrait (Snyder, Science 74:151, 1931; Levit and Soboleva, J. Genetics30:389, 1935; Blakeslee, Proc. Acad. Natl. Acad. Sci. USA 18:120, 1932;Lee, Ohio J. Science 34:337, 1934; Harris and Kalmus, Ann. Eugenics,London 15:24, 1949). Over time however, many reports emerged whichcontradicted this model (Falconer, Ann. Eugenics 13:211, 1946-47; Reddyand Rao, Genet. Epidemiol. 6:413, 1989; Olson et al., Genet. Epidemiol.6:423, 1989). Linkage studies have been equally conflicting. Initialstudies provided very strong support for linkage to the KEL blood groupantigen (later determined to reside on chromosome 7q3)(Chautard-Freire-Maia et al., Ann. Hum. Genet. 38:191, 1974; Conneallyet al., Hum. Hered. 26:267, 1976), but other studies failed to providesignificant support for this linkage (Spence et at, Hum. Genet. 67:183,1984). The only genome-wide linkage survey was performed with therelated compound propyl-thiouracil. This study produced evidence forlinkage to loci on chromosome 5p, and a suggestion of linkage to markerson chromosome 7q31, at a distance of ˜35 cM from KEL (Reed et al., Am.J. Hum. Genet. 64:1478, 1999).

We performed a genome-wide linkage analysis with the Utah C.E.P.H.families (Dausset et al., Genomics 6:575, 1990; NIH/CEPH CollaborativeMapping Group, Science 258:67, 1992; Materials and methods are availableas supporting material on Science Online.) using a blind sorting test tomeasure individual's PTC sensitivity thresholds (Materials and methodsare available as supporting material on Science Online; Harris andKalmus, Ann. Eugenics, London 15:24, 1949; Kalmus, Ann. Hum. Genet.22:222, 1958), and demonstrated strong support for a major locus onchromosome 7q, close to KEL (Prodi et al., Am. J. Hum. Genet. Suppl.71(4):464, 2002; Drayna et al., Hum. Genet. 112:567, 2003) with acritical region spanning approximately 4 Mb in the region of D7S661,with a maximum lod score of 8.85 (Drayna et al., Hum. Genet. 112:567,2003).

Bioinformatic analyses (Materials and methods are available assupporting material on Science Online.) indicated the ˜4 Mb region onchromosome 7q contains over 150 genes, including the KEL blood groupantigen, corning previous linkage studies (Chautard-Freire-Maia et al.,Ann. Hum. Genet. 38:191, 1974; 13. Conneally et al., Hum. Hered. 26:267,1976). In addition, this region contains a number of TAS2R bitter tastereceptor genes (Adler et al., Cell 100:693, 2000) and odorantreceptor-like genes (Buck and Axel, Cell 65:175, 1991). All TAS2R's (9genes) and OR-like genes (7 genes) were evaluated as candidates bysequencing the entire single coding exon, the 3′ UTR, and 300 bpupstream in individuals within families showing linkage to chromosome7q, and numerous sequence variants were observed (Ewing et al., GenomeRes. 8:175, 1998; 26. Gordon et al., Genome Res. 8:195, 1998. Seqman(DNA STAR, Madison, Wis.)). One of these variants demonstrated strongassociation with taste phenotype across different C.E.P.H. families(chi-square p<10⁻¹⁰), suggesting it may be the functional change orclose to the functional change(s). To more fully understand linkage andLD relationships in this region, we performed further analysis by meansof 50 SNPs at an average spacing of 50 kb across this interval. TheseSNPs revealed crossover breakpoints in the Utah C.E.P.H. families thatreduced the minimal region to 2.6 Mb.

Using these 50 SNP's, strong LD was observed between taster status andmarkers in only one portion of this 2.6 Mb interval. This was observedinitially in the chromosome 7-linked families (12 families containing107 individuals) and subsequently in unrelated non-tasters from both theC.E.P.H. sample (an additional 8 individuals) and in a secondreplication population (the NIH sample, 15 non-taster and 14 tasterCaucasians, 7 non-taster and 9 taster East Asians). Significant LD wasobserved across a 150 kb region, extending from approximately139,835,000 to 139,981,000 bp on the chromosome 7 genomic sequence(available on the Web at ncbi.nlm.nih.gov/genome/guide/human). In theNIH sample of 45 individuals, analysis of chi-square (equivalent to r²)and delta statistics showed clear peak values for each measure withinthe BAC RP11-707F14 (AC073647.9)(p<10⁻¹⁰), at the identical location inthe Caucasian and East Asian subgroups as well as for theMantel-Haenszel combined chi-square. In a group of 37 unrelatednon-taster individuals (12 Utah individuals and 25 individuals from theNIH sample who collectively had the poorest PTC sensitivities), thephysical distance over which these individuals carried unambiguoushaplotypes sharing the same SNP alleles extended an average of 61 kb,with the minimal shared region extending from 42,445 bp to 72,141 bp inthis BAC, a distance of 29,696 bp. Bioinformatic and gene predictionanalyses revealed that the only gene in this 29.7 kb interval was theTAS2R bitter receptor gene in which we originally identified strong LD.

This gene, which we have designated PTC, consists of 1002 bp in a singleexon, encoding a 7 transmembrane domain, G-protein-coupled receptor thatshows 30% amino acid identity with human TAS2R7, the most closelyrelated member of this family. Within this gene, we identified 3 commonSNPs, all of which result in amino acid changes in the protein (Table1). The A49P variant demonstrated a strong association overall withtaster status in the Utah sample (Table 2), and an even strongerassociation in the NIH replication sample (Table 2). The association oftaster status with the val262 allele was similarly strong in both theUtah and NIH samples (Table 2). To better understand the effect of theseSNP's, we investigated the haplotypes in this gene.

TABLE 1 Polymorphisms within the PTC gene Position (bp. a.a.) AlleleFrequency AA encoded 145  49 C .48 Pro G .51 Ala 785 262 C .38 Ala T .62Val 886 296 G .38 Val A .62 Ile

TABLE 2 The effect of homozygosity for SNPs on phenotype Homozygous No.of subjects (total no.) SNP Sample Non-tasters Tasters χ² P value Ala 49Utah 48 (51) 21 (129) 27.23 1.81 × 10⁻⁷  NIH 22 (23) 3 (61) 72.74 1.61 ×10⁻¹⁶ Val 262 Utah 38 (51) 14 (129) 23.40 1.10 × 10⁻⁶  NIH 21 (23) 0(61) 74.44 6.83 × 10⁻¹⁷ *The third SNP, I296V, was in complete linkagedisequilibrium with V262A (and thus gave identical results to V262A)except in one African-American subject.

Haplotype analysis in the Utah and NIH samples revealed two predominanthaplotypes at the three SNPs in this gene. Named in the order of thethree SNPs (A49P, V262A, and I296V), the non-taster haplotype AVI andtaster haplotype PAV accounted for 47% and 49% of all haplotypesrespectively in the European sample, and 30% and 70% respectively in theEast Asian sample. Europeans also possessed the presumed recombinanttaster haplotype AAV at a frequency of 3%. The haplotype associationwith taster status was more definitive than for individual SNP's; thestrongest association with non-taster status is for the AVI homozygote,followed by the compound heterozygote AVI/AAV (Table 3).

TABLE 3 Haplotype association with taste phenotypes No. of subjectsHaplotypes Sample Non-tasters Tasters AVI/AVI Utah 38 14 NIH 21 0AVI/AAV Utah 10 7 NIH 1 3 */PAV Utah 3 108 NIH 1 58 *indicates anyhaplotype found in the sample. No AAV homozygotes were observed ineither sample.

Due to the broad and continuous distribution of PTC sensitivity in thepopulation, we went on to analyze PTC scores as a quantitative trait.There was a consistent and significant difference in PTC scores betweendiplotypes in both the Utah and the NIH samples, consistent acrossracial groups. PAV homozygotes had the highest mean PTC scores (Utah:10.69, NIH: 10.00), PAV heterozygotes had slightly but significantlylower mean PTC scores (Utah: 9.65, NIH: 8.81) than the PAV homozygotes(Utah sample: χ²=8.41, p=0.0037, NIH replication sample: t=3.29,p=0.0017). AVI homozygotes had the lowest mean PTC scores (Utah: 4.31,NIH: 1.86). Thus the taster PAV form of the gene displays a heterozygoteeffect, with two copies conferring greater PTC sensitivity than a singlecopy. The difference in mean PTC score between the rare AAV/AVIheterozygotes and the AVI homozygotes was significant in the NIH sample(t=5.44, p=5.41×10⁻⁵) and tended toward significance in the Utah familysample (χ²=2.39, p=0.122). PAV/AAV heterozygotes were not significantlydifferent from PAV/AVI heterozygotes (χ²=0.58, p=0.45).

Differences in PTC score by diplotype in the Utah families were alsohighly significant in a multivariate analysis (χ²=148.95, p<10⁻³³). Sexand the haplotype effect explain 59.7% of the total variance in PTCscores. Analysis of variance of the NIH sample confirmed these results(F=152.73, p<10⁻³²), with 84.8% of the variance explained by thehaplotype effect. The differences were also significant in both theCaucasian subgroup of the replication sample (F=78.60, p<10⁻¹⁸) and theEast Asian subgroup (F=139.02, p<10⁻¹¹).

The bimodal distribution of PTC scores is a combination of theunderlying distributions of the PTC diplotypes, i.e. genotypes atmultiple variable sites with consideration of haplotype. The appearanceof bimodality is driven by the distribution of the common AVIhomozygote, PAV/AVI heterozygote and PAV homozygote diplotypes. The modeof inheritance of PTC taste sensitivity has been a subject ofcontroversy (Guo and Reed, Ann. Hum. Biol 28:111, 2001; Reddy and Rao,Genet. Epidemiol. 6:413, 1989; Olson et al., Genet. Epidemiol. 6:423,1989). To determine whether there was evidence for additional geneticcontributions to PTC score, we examined the heritability in subsets ofthe Utah sample. In the subgroups which were large enough to giveaccurate estimates, heritability was 0.26±0.19 (83 subjects in 20families) in the PAV/AVI subgroup, and 0.50±0.33 in the AVI/AVI subgroup(46 subjects in 17 families). The increase in heritability in the lossof function diplotype group (AVI/AVI) indicates that there may be othergenetic factors that interact with FTC and can restore some measure oftaste sensitivity in this group. For Caucasians and East Asians, ourresults are largely consistent with a model of a major recessive QTLmodified either by a polygenic (Reddy and Rao, Genet. Epidemiol. 6:413,1989) or single locus (Olson et al., Genet. Epidemiol. 6:423, 1989)residual background effect.

Due to the high frequency of the PAV and AVI haplotypes in thepopulation, we sought to determine which haplotype represents theoriginal form of the FTC gene. We sequenced this gene in 6 primatespecies: humans and one individual each from chimpanzee, lowlandgorilla, orangutan, crab-eating macaque (an old world monkey), andblack-banded spider monkey (a new world monkey), representing over 25million years of evolutionary divergence. All of the non-human primateswere homozygous for the PAV form, indicating that the AVI form arose inhumans after the time they diverged from the nearest common primateancestors.

Five different haplotypes were observed worldwide (Table 4). InEuropeans and Asians, the taster haplotype PAY and the non-tasterhaplotype AVI make up the vast majority of haplotypes present. Twoadditional haplotypes, PVI and AAI, were observed only in individuals ofsub-Saharan African ancestry, consistent with other reports of increasedgene haplotype diversity in this population (Stephens et al., Science293:489, 2001). The common non-taster AVI haplotype was observed in allpopulations except Southwest Native Americans, who were exclusivelyhomozygous for the PAV haplotype, consistent with the reported lowfrequency of non-tasters in this population (Guo and Reed, Ann. Hum.Biol. 28:111, 2001). Thus overall, the worldwide distribution of thesehaplotypes is consistent with the large anthropologic literature on thedistribution of this phenotype (Boyd, “Genetics & the Races of Man. Anintroduction to modem physical anthropology.” Little Brown and Company,Boston, 1950; Tills et al., “The Distribution of Human Blood Groups andother Polymorphisms,” Supplement, 1^(st) Edition. Oxford UniversityPress, Oxford, 1983).

TABLE 4 Frequency of PTC Gene Haplotypes in Populations Worldwide S.W.Native European West Asian East Asian African American Haplotype (n =200) (n = 22) (n = 54) (n = 24) (n = 18) AVI 0.47 0.67 0.31 0.25 AAV0.03 0.04 AAI 0.17 PAV 0.49 0.33 0.69 0.50 1.00 PVI 0.04

The amino acid substitutions in the PTC protein may affect the functionof this protein in several ways. Position 49 resides in the predictedfirst intracellular loop, and this SNP represents a major amino acidalteration, proline in tasters to alanine in non-tasters. The SNP's atpositions 262, in the predicted 6^(th) transmembrane domain, andposition 296, in the predicted 7^(th) transmembrane domain specifyrelatively conserved amino acid changes, alanine to valine and valine toisoleucine, respectively. Based on phenotype data, we hypothesize thatthe substitutions at positions 49 and 262 significantly alter thebiochemical function of this protein, while the substitution at position296 modifies the function more subtly. These alterations could affectcoupling to its cognate G proteins on the intracellular side of theplasma membrane, as has been observed for other variants in the firstintracellular loop (Nabhan et al., Biochem. Biophys. Res. Comm.212:1015, 1995; O'Dowd et al., J. Biol. Chem. 263:15985, 1988), or inother portions of these proteins (G protein receptor database: availableon the Web at gpcr.org, grap.fagmed.uit.no). Given that PTC and othercompounds which contain the N—C═S moiety are both bitter and toxic inlarge doses, it will be of interest to determine how the non-tasterallele rose to such high frequency, especially in the Europeanpopulation.

Substantial variation in taste sensitivity exists in humans (Blakesleeand Salmon, Proc. Natl. Acad. Sci. USA 21:84, 1935), and given the greatdegree of sequence diversity and variation in bitter taste receptorgenes (Ueda et al., Biociem. Biophys. Res. Comm. 285:147, 2001), wehypothesize much of this phenotypic variation is genetic in origin.Understanding the nature of this variation, especially variation inbitter taste, and its relationship to diet and other behaviors such assmoking may have important implications for human health (Tepper, Am. J.Hum. Genet. 63:1271, 1998; Enoch et al., Addictive Behav. 26:399, 2001).

Example 2 Natural Selection and Molecular Evolution in PTC

This example describes an investigation of selective effects on thephenylthiocarbamide (PTC) gene by use of analyses of molecular geneticdata. By examining patterns of DNA sequence variation, we were able totest the PTC gene for evidence of long-term selective pressures. Aspredicted by Fisher >60 years ago (Fisher et al., Nature 144:7-50,1939), we found support for the hypothesis that balancing naturalselection has acted to maintain taster and nontaster alleles in humanpopulations. This investigation has also been reported in Wooding et al.(J. Hum. Genet. 74:637-646, 2004), which is incorporated herein byreference in its entirety.

The ability to taste PTC is a classic phenotype that has long been knownto vary in human populations. This phenotype is of genetic,epidemiologic, and evolutionary interest because the ability to tastePTC is correlated with the ability to taste other bitter substances,many of which are toxic. Thus, variation in PTC perception may reflectvariation in dietary preferences throughout human history and couldcorrelate with susceptibility to diet-related diseases in modernpopulations.

To test R. A. Fisher's long-standing hypothesis that variability in PTCperception has been maintained by balancing natural selection, weexamined patterns of DNA sequence variation in the recently identifiedPTC gene, which accounts for up to 85% of phenotypic variance in thetrait. We analyzed the entire coding region of PTC (1,002 bp) in asample of 330 chromosomes collected from African (n=62), Asian (n=138),European (n=100), and North American (n=20) populations by use of newstatistical tests for natural selection that take into account thepotentially confounding effects of human population growdh. Twointermediate-frequency haplotypes corresponding to “taster” and“nontaster” phenotypes were found. These haplotypes had similarfrequencies across Africa, Asia, and Europe. Genetic differentiationbetween the continental population samples was low (FST=0.056) incomparison with estimates based on other genes. In addition, Tajima's Dand Fu and Li's D and F statistics demonstrated a significant deviationfrom neutrality because of an excess of intermediate-frequency variantswhen human population growth was taken into account (P<0.01). Theseresults combine to suggest that balancing natural selection has acted tomaintain “taster” and “nontaster” alleles at the PTC locus in humans.

Methods

DNA sequences from the entire coding region of PTC (1,002 bp, 333 aminoacids) were obtained by use of methods described above and in the workof Kim et al. (Science 299:1221-1225, 2003) and Drayna et al. (Hum Genet112:567-572, 2003) (both of which are incorporated herein by referencein their entireties), from 165 individuals of the following descents:African (9 sub-Saharan Africans from Coriell Human Variation panel HD12,22 Cameroonians), Asian (17 Chinese, 13 Japanese, 12 Koreans, 7 MiddleEasterners, 10 Pakistanis, 10 other Southeast Asians), European (10Hungarians, 45 Utah samples from Centre d'Etude du PolymorphismeHumain), and North American (10 Southwest Native Americans). Forcomparison, sequences were also obtained from one chimpanzee (Pantroglodytes) and one gorilla (Gorilla gorilla).

Ambiguous haplotypes were resolved using molecular techniques. In suchindividuals, the two allelic versions of the gene were cloned as singlePCR products and individual clones were sequenced to reveal bothhaplotypes. Phylogenetic relationships among haplotypes were inferredusing the neighbor-joining algorithm of the PHYLIP software package(Felsenstein 1993). This tree, which was rooted using the gorillasequence, was then used to determine the polarity of character states.Evolutionary relationships among haplotypes were visualized using aminimum spanning tree generated by the ARLEQUIN computer program(Schneider et al., “ARLEQUIN version 2.000: a software for populationgenetics data analysis. Genetics and Biometry Laboratory, Department ofAnthropology, University of Geneva, Geneva, Switzerland 2000).

Tajima's D (Tajima, Genetics 123:585-595, 1989) and Fu and Li's D and Fstatistics (Fu and Li, Genetics 133:693-709, 1993) were used to test thehypothesis that patterns of diversity in humans are consistent with thehypothesis of neutrality. To avoid confusion, we refer to Tajima's D as“DT” and Fu and Li's D as “DF.” These tests were performed by simulating10,000 gene genealogies and comparing statistics obtained from thesimulations with the observed statistic, as described by Tajima(Genetics 123:585-595, 1989) and Fu and Li (Genetics 133:693-709, 1993).To incorporate varying assumptions about population size change in humanpopulations, these simulations were performed using the algorithm ofRogers (Evolution 49:608-615, 1995). This algorithm assumes that humanpopulation sizes increased suddenly from an ancient population size (N0)to a larger population size (N1), t generations ago, with infinite-sitesmutation rate. Patterns of genetic diversity produced under theseconditions approximate those produced under more complicated conditions,such as exponential and logistic growth (Wooding & Rogers, Genetics161:1641-1650, 2002).

Tests for excesses of synonymous and nonsynonymous nucleotidesubstitutions were performed using the methods of McDonald and Kreitan(Nature 351:652-654, 1991) and Li et al. (Mol Biol Evol 2:150-174,1985). The McDonald-Kreitman test (McDonald & Kreitman, Nature351:652-654, 1991) uses a Fisher's exact test to determine whether theratio of synonymous and nonsynonymous substitutions differs between twocategories: polymorphisms that are variable within species andpolymorphisms that distinguish species (i.e., fixed differences). Weused the McDonald-Kreitman test to examine polymorphisms found in humansand chimpanzees. The KA/KS test determines whether there is an overallexcess of synonymous or nonsynonymous nucleotide substitutions (Li etal., Mol Biol Evol 2:150-174, 1985).

Tests for genetic differentiation between populations were performedusing Slatkin's linearized FST statistic (Slatkin, Genet Res 58:167-175,1991). The statistical significance of these values was assessed usingthe bootstrap method of Excoffier et al. (Excoffier et al., Genetics131:479-491, 1992), in which observed values are compared with FSTvalues simulated by randomly allocating chromosomes to differentpopulations. These tests used 10,000 bootstrap replications.

Results and Discussion

DNA sequencing revealed five variable nucleotides in humans, as detailedin Example 1. These variants were partitioned into seven haplotypes. Thechimpanzee and gorilla were both homozygous at all nucleotide positionsand thus carried one haplotype each. Human and chimpanzee sequencesdiffered by an average of 8.3 nucleotides, as did human and gorillasequences. The chimpanzee and gorilla sequences differed by sixnucleotides. In Table 5A, each haplotype is summarized in two rows. Thetop row summarizes nucleotide variation in the haplotype, and the bottomrow summarizes amino acid variation in the haplotype. Each columnrepresents a codon containing a variable nucleotide position, which isindicated at the top of the column. Shaded columns indicate the threevariable amino acid positions used for haplotype designation in Example1 and Kim et al. (Science 299:1221-1225, 2003). The number ofoccurrences of each haplotype is indicated in Table 5B, for the African(Af), Asian (As), European (Eu), and North American (NA) samples.Haplotype counts are not given for the chimpanzee and gorilla haplotypes(ptA and ggA, respectively), which were each observed twice.

TABLE 5A Variable Nucleotide Positions in PTC Haplotypes.

¹SEQ ID NO: (NA/AA)—Sequence ID number for Nucleic Acid/(Amino Acid.

TABLE 5B Occurrences of Variable Nucleotides in Human PTC Haplotypes. AfAs Eu NA Total Haplotype hsA 38 76 51 19 184 hsB 1 0 0 0 1 hsC 1 0 0 0 1hsD 9 1 0 0 10 hsE 2 0 5 0 7 hsF 1 0 0 0 1 hsG 10 61 54 1 126 62 138 11020 330 Total

All five nucleotide substitutions observed in humans caused amino acidsubstitutions, as did three of the six substitutions distinguishinghuman and chimpanzee. The observed rate of nonsynonymous substitutionwas substantially higher than is usually observed in human genes,suggesting that positive natural selection may have acted to preservenew nonsynonymous variants (Makalowski & Boguski, Proc Natl Acad Sci USA95:9407-9412, 1998; Yang et al., Trends Ecol Evol 15:496-503, 2000;Nekrushenko et al., Genome Res 12:198-202, 2001; Bamshad and Wooding,Nat Rev Genet 4:99-111, 2003). To test the hypothesis that an excess ofnonsynonymous substitutions was present, we first analyzed the human andchimpanzee sequences by use of a McDonald-Kreitman test (Nature351:652-654, 1991), as described in the “Methods” section. This testshowed that the excess of nonsynonymous substitutions observed in humanswas not statistically significant (P>0.10). A KA/KS test showed that theoverall ratio of synonymous to nonsynonymous substitutions did notdiffer significantly from expectation under neutrality (P>0.10). Thesenonsignificant results may be attributable to the low number ofpolymorphisms observed, which weakens the tests. Thus, although notablefor being higher than in most genes, the bias toward nonsynonymousvariants in our sample was not sufficient to reject the hypothesis ofneutrality.

The minimum spanning tree revealed that the human sample was dominatedby two major haplotypes, hsA and hsG, differing by three amino acidsubstitutions. These two haplotypes, which account for >90% of sampledchromosomes, are strongly associated with taster (hsA) and nontaster(hsG) status, respectively (Drayna et al., Hum Genet 112:567-572, 2003;Kim et al., Science 299:1221-1225, 2003). In addition, the hsA and hsGhaplotypes were both found at intermediate frequencies: 0.55 and 0.38. Avariety of factors, including population subdivision and balancingnatural selection, can lead to the presence of two or moreintermediate-frequency haplotypes in gene genealogies (Marjoram &Donnelly, Genetics 136:673-683, 1994; Bamshad and Wooding, Nat Rev Genet4:99-111, 2003). The evolution of two or more intermediate-frequencyclusters is also surprisingly common under selectively neutralconditions (Slatkin & Hudson, Genetics 129:555-562, 1991).

To test whether patterns of DNA sequence variation in PTC fitexpectations under the hypothesis of evolutionary neutrality, weanalyzed the sequences by use of the DT, DF, and F statistics. Thesestatistics are functions of the number of variable nucleotide positionsin a sample of sequences, the mean pairwise difference betweensequences, and the number of derived variants that are only observedonce in the sample, all of which are affected by natural selection(Tajima, Genetics 123:585-595, 1989; Fu and Li, Genetics 133:693-709,1993). For example, positive natural selection leading to the rapidfixation of a single, advantageous haplotype will result in a decreasein the expected number of polymorphic sites, a decrease in the meanpairwise difference between sequences, and an increase in the number ofvariants observed only once in the sample (Fu and Li, Genetics133:693-709, 1993). In contrast, balancing natural selection can lead toan increase in all three of these values (Fu and Li, Genetics133:693-709, 1993).

Tests of these statistics performed under the standard assumption ofconstant population size failed to reject the hypothesis of neutralityin PTC (DT=1.55, P>0.05; DF=−1.46, P>0.90; F=−0.50, P>0.60). However,several lines of evidence based on archaeology, genetics, andlinguistics suggest that human populations have grown dramatically(>100-fold) over the past 100,000 years (Ruhlen, “The origin oflanguage.” John Wiley and Sons, New York 1994; Klein, “The human career:human biological and cultural origins.” University of Chicago Press,Chicago 1999; Stiner et al., Science 283:190-194, 1999; Excoffier. CurrOpin Genet Dev 12:675-682, 2002). Such growth is known to have strongeffects on genetic diversity (Rogers & Harpending, Mol Biol Evol9:552-569, 1992). For example, diversity patterns in populations thathave grown are often characterized by an excess of low-frequency geneticvariants and a low mean pairwise difference between sequences, both ofwhich lead to reductions in the expected values of all three of thestatistic we tested (Wooding & Rogers, Genetics 161:1641-1650, 2002).Given evidence for population increase in the Upper Pleistocene and thepossible effects of this growth on patterns of genetic diversity, theassumption of constant population size is likely inappropriate.

To investigate the possibility that incorrect assumptions aboutpopulation history were causing a type II statistical error (i.e., afailure to reject the null hypothesis of neutrality when it is false) inour initial tests, we devised new tests of the DT, DF, and F statisticsthat take population growth into account. These tests were performed asdescribed in the “Methods” section, under the assumption that humanpopulations increased suddenly from an ancient effective population sizeof 10,000 to a larger effective population size, N1, t years beforepresent, with a nucleotide-substitution rate of 10-9/site/year. Thesevalues are representative of those inferred for nuclear genes in humans(Tishkoff & Verrelli, Annu Rev Genomics Hum Genet 4:293-340, 2003).Because there is some disagreement about the timing and magnitude ofthis expansion (Hey, Mol Biol Evol 14:166-172, 1997; Fay and Wu, MolBiol Evol 16:1003-1005, 1999; Harris and Hey, Evol Anthropol 8:81-86,1999; Hey and Harris, Mol Biol Evol 16:1423-1426, 1999; Harpending andRogers, Annu Rev Genomics Hum Gent 1:361-385, 2000; Wall & Przeworski,Genetics 155:1865-1874, 2000; Excoffier. Curr Opin Genet Dev 12:675-682,2002; Ptak & Przeworski, Trends Genet 18:559-563, 2002), we iterativelytested the DT, DF, and F statistics for population histories withmagnitudes of population growth from 1-fold to 1,000-fold and dates ofpopulation expansion from 0 to 200,000 years ago.

This procedure revealed that tests of all three statistics are highlysensitive to assumptions about population growth. For example, theassumption of 100-fold growth 100,000 years ago resulted in a change ofDT's P value from 0.07 to 0.01. CIs generated for the DT statisticshowed that the hypothesis of neutrality was rejected (at a two-tailed Pvalue cutoff of 0.025) under all population histories in which the humanpopulation expanded between 15-fold and 1,000-fold between 10,000 and200,000 years ago. In addition, under the population history parametersfor which observed DT values differed significantly from expectation,the values were greater than expected. Thus, our data departed fromexpectation in a direction consistent with the hypothesis of balancingnatural selection (Tajima, Genetics 123:585-595, 1989; Fu and Li,Genetics 133:693-709, 1993). Results were similar for CIs generatedusing the DF and F statistics, which rejected the hypothesis ofneutrality for all population histories in which the human populationexpanded between 15-fold and 1,000-fold between 30,000 and 200,000 yearsago. Thus, the hypothesis of neutrality in PTC was rejected by thesetests under all but the most conservative assumptions about populationgrowth in humans.

The sensitivity of the DT, DF, and F statistics to population growth hasimplications beyond the detection of natural selection in PTC. All threeof these statistics are widely used in tests for natural selection inhumans, usually under the assumption that human population sizes haveremained constant (Tishkoff & Verrelli, Annu Rev Genomics Hum Genet4:293-340, 2003). As we have shown, this assumption is highlyconservative in the detection of balancing natural selection. However,the assumption of constant population size is anticonservative in thedetection of positive natural selection, which leads to reductions indiversity nearly identical to those caused by population growth (Tajima,Genetics 123:585-595, 1989; Fu and Li, Genetics 133:693-709). For thisreason, tests for positive natural selection that use the DT, DF, and Fstatistics are vulnerable to type I statistical errors (i.e., therejection of the null hypothesis when it is true) if human populationincreases are not taken into account. With this in mind, thesignificance of many earlier tests of the DT, DF, and F statistics inhumans, including our own (e.g., Wooding & Rogers, Hum Biol 72:693-695,2000; Wooding et al., Am J. Hum Genet 71:528-542, 2002, may need to bereconsidered.

Balancing selection is not the only force that can lead to significantlyhigh DT, DF, and F values. Such patterns can also be caused bypopulation subdivision, which allows the persistence of divergenthaplotypes in different geographical regions Kaplan et al., Genet Res57:83-91, 1991; Hudson et al., Mol Biol Evol 9:138-151, 1992; Wakeley,Theor Popul Biol 59:133-144, 2001; Laporte & Charlesworth, Genetics 162:501-591, 2002). In our analyses, two sources of population subdivisionwere potentially important: subdivision between continents andsubdivision within Africa.

Population subdivision between continents is not large (Tishkoff &Verrelli, Annu Rev Genomics Hum Genet 4:293-340, 2003), but it could besufficient to confound statistics like DT, DF, and F. To test thehypothesis that the presence of the two intermediate-frequencyhaplotypes in our data is the result of subdivision between continents,we analyzed patterns of genetic differentiation among continentalpopulations by use of the FST statistic, which compares the level ofgenetic diversity within subpopulations to levels of diversity in thepopulation as a whole (Hartl and Clark, Sinauer Associates, Sunderland,Mass., 1997). In our data, diversity patterns were driven largely by thefrequencies of the hsA and hsG haplotypes, which were present at similarfrequencies in most populations. The FST value observed among all fourcontinental samples was 0.056. This value is significantly differentfrom zero (P<0.025) but is lower than is typically observed in nucleargenes, which generally have values of 0.15 (Przeworski et al., TrendsGenet 16:296-302, 2001; Schneider et al., Mech Ageing Dev 124:17-25,2003; Tishkoff & Verrelli, Annu Rev Genomics Hum Genet 4:293-340, 2003;Watkins et al., Genome Res 13:1607-1618, 2003). This FST value is lowerthan 80% of those reported for 25, 549 SNPs by Akey et al. (Genome Res12:1805-1814, 2002), for instance, and is also lower than 45% of thosereported for 1, 627 genes by Schneider et al. (Mech Ageing Dev124:17-25, 2003). The latter sample would be expected to haveexceptionally low FST values because it included a large number ofindividuals from admixed populations, such as African-Americans andHispanic-Latinos. The FST observed in our sample suggests thatcontinental populations are less different with respect to variation inPTC than they are with respect to most other genes, not more differentas would be expected if population subdivision or local adaptation hadoccurred.

Between-continent FST values were strongly affected by the inclusion ofthe North American sample, owing to the very high frequency of the hsAhaplotype (0.95) in that group. The FST value excluding the NorthAmerican sample was substantially lower than for the sample as a whole:0.025. This value is significantly greater than zero (P<0.025) but islower than 75% of FST values reported by Schneider et al. (Mech AgeigDev 124:17-25, 2003) and lower than 90% of values reported by Akey etal. (Genome Res 12:1805-1814, 2002). The strong effect of the NorthAmerican sample could be due to a variety of factors. First, our NorthAmerican sample is small and may not provide an accurate representationof genetic diversity in North Americans. Estimates of the frequency ofnontaster alleles in larger North American samples vary widely(Cavalli-Sforza et al., “The history and geography of human genes,”Princeton University Press, Princeton, 1994). Second, archaeological andlinguistic evidence suggest that North America was not inhabited byhumans until recently (15,000 years ago) (Evol Anthropol 8:208-227,1999; Nettle, Proc Natl Acad Sci USA 96:3325-3329, 1999), and geneticevidence suggests that North and South American populations descendedfrom a relatively small number of founders that entered the Americas viathe Bering Strait (Torroni et al., Am J Hum Genet 53:563-590, 1993;Torroni et al., Am J Hum Genet 53:591-608, 1993b). Both of these factorscan have strong effects on FST values (Urbanek et al., Mol Biol Evol13:943-953, 1996).

Evidence for extensive subdivision has also been found within Africa(Schneider & Excoffier, Proc Natl Acad Sci USA 96:10597-10602, 1999;Tishkoff & Williams, Nat Rev Genet 3:611-621, 2002; Yu et al., Genetics161:269-274, 2002). As with continental subdivision, subdivision withinAfrica could inflate DT, DF, and F statistics, yielding a falsesignature of balancing natural selection. To test the hypothesis thatsubdivision within the African sample in our study was responsible forthe high observed values of these statistics in our sample as a whole,we performed separate DT, DF, and F tests for each continent under theassumptions of (1) no growth and (2) 100-fold growth 100,000 years ago.As shown in Table 6, these statistics were significantly higher thanexpected in Asia and Europe, even when population growth was not takeninto account. Furthermore, the P values of the DT, DF, and F statisticsin the African population alone were greater than for the sample as awhole, not lower as would be expected if subdivision within Africa werecausing the presence of high overall D values. Thus, substructure inAfrican populations cannot solely explain the high DT values observed inPTC.

TABLE 6 Results of Statistical Tests OBSERVED VALUE P VALUE FOR FORSTATISTIC No Growth Growth SAMPLE D_(r) D_(F) F D_(T) D_(F) F D_(T)D_(F) F Africa .46 −.89 −.56 .18 .80 .56 .01 .03 .01 Asia 2.94 −.67 .57.01 .76 .28 .01 .01 .01 Europe 2.91 −.62 .59 .01 .81 .28 .01 .01 .01North America −2.66 −2.58 −3.05 .99 .99 .68 .99 .91 .87 All 1.55 −1.46−.50 .08 .90 .64 .01 .01 .01 P values given are the fraction ofsimulations that yielded a greater value than observed. The “Growth”columns show P values calculated under the assumption that, 100,000years ago, the human population expanded 100-fold.

Taken together, three lines of evidence suggest that balancing naturalselection has acted to maintain high levels of diversity in humanpopulations. First, two haplotypes strongly associated with functionallydivergent phenotypes dominate the sample. Second, under reasonableassumptions about human population history, the distribution ofpolymorphism frequencies in our sample has significantly moreintermediate-frequency variants than expected under neutrality (P<0.01).Third, the geographical distribution of the taster and nontaster allelesis not consistent with the hypothesis that they have arisen throughpopulation subdivision within or between continents. Thus, R. A.Fisher's hypothesis that balancing natural selection has maintainedtaster and nontaster alleles appears to hold true in humans (Fisher etal., Nature 144:7-50, 1939).

Evidence for balancing selection at the PTC locus does not imply thatother selective pressures have been absent. For instance, it is possiblethat positive natural selection led to the rapid evolution of thenontaster allele, which was then maintained by balancing selection. Thispossibility might explain the unusually large number of nonsynonymousnucleotide substitutions found in this gene. It is also possible thatspecific PTC alleles have been favored by positive natural selection inparticular environments, resulting in local adaptation. Such effectsmight account for the high frequency of PTC taster alleles in New Worldpopulations and the significant low DT, DF, and F in our North Americansample.

The mechanism through which balancing natural selection has maintaineddivergent PTC alleles in human populations remains unclear. No stopcodons or deletions, which might yield nonfunctional alleles, have yetbeen found at the PTC locus. In addition, although several haplotypesare present in our sample, two account for >90% of observations: hsA andhsG. If nontaster alleles were simply “broken” taster alleles, it seemslikely that a greater diversity of nontaster alleles would be expected(Harding et al., Am J. Hum Genet 66:1351-1361, 2000). One possibility isthat PTC heterozygotes gain a fitness advantage through the perceptionand avoidance of a larger repertoire of bitter toxins than homozygotes.We currently believe that PTC nontaster alleles may encode functionalreceptor molecules that bind to toxic bitter substances other than PTC.

Example 3 Identification of SNPs in Other T2R Bitter Taste Receptors

Common allelic variants of a member of the TAS2R bitter taste receptorgene family underlie variation in the ability to tastephenylthiocarbamide (PTC). To extend these results to other bitterreceptors, we have sequenced 22 of the 24 known TAS2R genes in a seriesof populations worldwide, including Hungarians, Japanese, Cameroonians,Pygmies, and South American Indians. This example provides descriptionof this analysis, which was used to generate a comprehensive collectionof single nucleotide polymorphisms in human T2R putative bitter tastereceptors.

Using conventional methods, members of the human T2R family of putativebitter taste receptors were analyzed for the presence of SNPs. AU SNPswere identified and analyzed by DNA sequencing. Genomic DNA encodingeach receptor was PCR'd using standard methods, and the products cyclesequenced with dye terminators using a Big Dye terminator kit from ABI.Products of the sequencing reactions were analyzed on an ABI 3730x1 DNAAnalyzer using the manufactures' recommendations. Other sequencingtechniques would be equally applicable to detecting SNPs in these genes.

The results of the comprehensive sequencing are presented in FIG. 1 andTable 7; specific individual variants are also described in the attachedSequence Listing. FIG. 1 shows, in addition to those SNPs confined oridentified by sequencing reaction, all SNPs found in dbSNP for thesegenes. See also Tables 7A, 7B, and 7C, below, which show the cSNPs andassociated haplotypes.

All 22 T2R genes contain common SNP's within their coding sequence, andwe identified an average of 4 A SNP's per T2R gene. Fifteen variantslisted in dbSNP were not observed to be polymorphic in our sample.However, many novel SNPs were identified; these are indicated with the“new” designation in FIG. 1. Of the SNP's we observed FIG. 1), 77% causean amino acid substitution in the encoded receptor protein, giving riseto a very high degree of receptor protein variation in the population.Four SNP's specify one allele that introduces an in-frame stop codon inthe gene. Some of the SNP's were observed only in individuals ofsub-Saharan African origin, and overall African samples displayed higherdiversity of alleles. This is consistent with the view that the majorityof human genetic variation resides within older African populations, anda fraction of this variation emerged and subsequently spread across theremainder of the world.

Example 4 Worldwide Coding Sequence Variation in Human Bitter TasteReceptors

This example describes the comprehensive evaluation of the worldwidevariation in the human bitter taste receptor gene repertoire, anddemonstrates that these genes exhibit a high degree of coding sequencediversity. On average these genes contain 4.2 variant amino acidpositions, and in aggregate, the 22 genes analyzed specify 109 differentprotein coding haplotypes. To investigate the effects of naturalselection on the bitter taste receptor genes overall, neutrality testswere performed sing Tajima's D statistic. Although none of theindividual D values departed significantly from expectation, the mean Dvalue was significantly higher than expectation, suggesting eitherbalancing natural selection or population subdivision has affected thefrequency of different alleles of these genes. In addition, the mean FSTvalue was significantly greater than expected, indicating that the highD values are attributable to differences between, not within,populations. Unlike the phenylthiocarbamide receptor, which showsevidence of strong balancing selection, other human bitter receptorgenes appear to be influenced by local adaptation. It is proposed thatthese genes have adapted under natural selection imposed by toxic bittersubstances produced by plants.

The human T2R38 (PTC) gene has been shown to exist in seven differentallelic forms although only two of these, designated the major tasterform and the major non-taster form, exist at high frequency outside ofsub-Saharan Africa (Example 1 and 2; Wooding, et al., Am. J. Hum. Genet.74:637-646, 2004). These two forms have been shown to be maintained bybalancing natural selection, and it has been suggested that thenon-taster form serves as a functional receptor for some other bittersubstance not yet identified. T2R38 (PTC) studies suggest that there maybe substantial additional complexity in the task of identifying specificligands for each bitter taste receptor, as different alleles of eachgene may encode receptors that recognize different ligands. Tofacilitate the resolution of this problem, we here identify all of thecommon variants and haplotypes of the nearly complete bitter receptorgene repertoire in humans, and examine population genetic aspects ofthis variation worldwide. All of the SNPs in T2R43 (see Example 3 andFIG. 1) were found in Cameroonian random individuals. Because parentalgenotypes were not available for these samples, the haplotypes for T2R43were not determined in this study.

Materials And Methods

Population samples: Human genomic DNA was obtained from 55 unrelatedindividuals in 5 different geographic populations including 21Cameroonians, 10 Amerindians, 10 Japanese, 9 Hungarians, and 5 Pygmies.All DNA samples except Cameroonian were provided by Coriell CellRepositories.

PCR and DNA sequencing: We sequenced the open reading frame (ORF) of 21out of 23 human T2R genes and combined this information with data fromthe T2R38 (PTC) gene published previously (Wooding et al., Am. J. Hum.Genet. 74:637-646, 2004). Primers for PCR amplification and forsequencing were designed (using software at the Primer3 Web site) toamplify the entire ORF of each T2R gene in humans.

PCR was performed in a total volume of 25 μl, containing 0.2 μM of eachdeoxynucleotide (Invitrogen), 15 pmol of each forward and reverseprimers, 1.0-1.5 mM of MgC12, 10 mM Tris-HCl (pH8.3), 50 mM KCl, 0.75 Uof Taq DNA polymerase (PE Biosystems), and 100 ng of genomic DNA. PCRconditions (PE9700, PE Biosystems) were as follows: 35 cycles ofdenaturation at 94° C. for 30 sec; annealing at 55° C. or 57° C.,depending on the primers for 30 sec; and extension at 72° C. for 1 min.The first step of denaturation and the last step of extension were 95°C. for 2 min and 72° C. for 10 min, respectively. Five microliters ofthe PCR products were separated and visualized in a 2% agarose gel.Fifteen microliters of this PCR product were then treated with 0.3 U ofshrimp alkaline phosphatase (USB) and 3 U of exonuclease I (USB) at 37°C. for 1 hr, followed by incubation at 80° C. for 15 min. This wasdiluted with an equal volume of dH₂O, and 6 μl was used for the finalsequencing reaction. Sequencing reactions were performed in bothdirections on the PCR products in reactions containing 5 pmol of primer,1 μl of Big Dye Terrminator Ready Reaction Mix (PE Biosystems), and 1 μlof 5× dilution buffer (400 mM Tris-HCl, pH 9 and 10 mM MgCl₂). Cyclingconditions were 95° C. for 2 min and 35 cycles of 94° C. for 20 sec, 55°C. for 20 sec, and 60° C. for 4 min. Sequencing reaction products wereethanol precipitated, and the pellets were resuspended in 10 μl offormamide loading dye. An ABI 3730 DNA sequencer was used to resolve theproducts, and data was analyzed by using ABI Sequencing Analysis (v.5.0) and LASERGENE-SeqMan software.

Data Analysis: Linkage disequilibrium between pairs of SNPs as estimatedusing Lewontin's disequilibrium statistic, D' (Lewontin, Genetics50:757-782, 1964). D' values were calculated using the GOLD softwarepackage (Abecasis, et al., Bioinformatics 16:182-183, 2000). Haplotypeand recombination rates between SNP pairs were estimated using the PHASE2.0.2 computer program (Stephens, et al., Am. J. Hum. Genet. 68:978-989,2001; Stephens, et al., Am. J. Hum. Genet. 73:1162-1169,2003), whichimplements the methods of Li and Stephens (Genetics 165:2213-2233,2003). This method uses likelihood-based algorithms to estimate abaseline rate of recombination across a region, as well as relativerates of recombination among SNP pairs. Recombination rates wereestimated for the two bitter taste receptor gene clusters on chromosomes7 and 12.

Neutrality tests were conducted using Tajima's D statistic, whichcompares the mean pairwise difference between randomly chosen sequencesin a sample with the number of segregating sites (Tajima, Genetics123:585-595, 1989). We tested the hypothesis of selective neutralityunder both the assumption of constant population size and the assumptionof 100-fold growth, 100,000 years ago, as described by Wooding et al.(Am. J. Hum. Genet. 74:637-646, 2004).

The distribution of Tajima's D values in our sample was tested todetermine whether the mean D value in bitter taste receptor genes wassignificantly greater than expected. This test was performed bysimulating 10,000 sets of 21 genes and comparing the mean D value ofeach simulated dataset with the mean D of the 21 genes in the observeddataset with theoretical expectations under both assumptions. Also, wecompared the mean D value observed in the 21 bitter taste receptor geneswith empirical expectations based on a set of 160 environmentallyresponsive genes reported by the NIH environmental Genome Project (EGP).This test was performed by randomly selecting 10,000 subsets of 21 genesfrom the EGP dataset and comparing the mean D value of each randomsubset with the mean D in the 21 observed bitter taste receptor genes.The fraction of comparisons in which the D calculated from our dataexceeded that in the simulated genes was treated as a one-tailedP-value.

FST values were calculated using the method of Slatkin (Genetics127:627-629, 1991), with continental regions defining subpopulations.Each observed FST value was tested to determine whether it wassignificantly different from zero using the method of (Excoffier, Curr.Opin. Genet. Dev. 12:675-682, 2002). This method compares the observedvalue of FST with values simulated by randomly allocating individualchromosomes to subpopulations. These tests used 10,000 bootstrapreplications.

The distribution of FST values in the bitter taste receptor genes wasalso tested to determine whether the mean FST in our data wassignificantly greater than the mean FST of 25,549 SNPs analyzed by Akeyet al., which were assembled from The SNP Consortium (TSC) allelefrequency project (snp.cshl.org/allele₁₃ frequency₁₃ project) (Akey etal., Genome Res. 12:1805-1814, 2002). These means were compared asfollows. First, one random SNP was chosen from each bitter tastereceptor gene to form a dataset composed of 21 SNPs. Next, 21 randomSNPs were chosen from the TSC dataset. Finally, the FST valuescalculated for each randomized dataset were compared. This procedure wasrepeated 10,000 times. The fraction of comparisons in which the FSTcalculated from our data exceeded that calculated from the TSC data wastreated as a one-tailed P-value.

Result and Discussion

We sequenced 21 human T2R (also known as TAS2R) genes to find codingregion single nucleotide polymorphisms (cSNPs) using 5 differentpopulations. These genes displayed a very high degree of nucleotidevariation ranging from one cSNP in T2R13 to 12 cSNPs in T2R48. Combinedwith previous results from T2R38 (PTC) gene (Wooding, et al., Am. J.Hum. Genet. 74:637-646, 2004), we identified a total of 127 cSNPs in thenearly complete set of T2R genes, with an average of six cSNPs per thegene (detailed in Table 7A, 7B, and 7C). For 32% of cSNPs, the minorallele was observed only once, suggesting they are not common. TheCameroonian population displayed the greatest number of these rarealleles, consistent with the view that African populations harbor higherlevels of diversity than do other populations. The remainder of thesecSNPs were approximately evenly divided into two classes; 36.2% had aminor allele frequency between 1% and 20%, and the remaining 31.5% hadminor allele frequencies between 20% and 50%. FIG. 2 shows thedistribution of sharing of cSNPs across these populations. ExcludingPygmies, four populations shared 25 cSNPs although only six cSNPs existin all the populations studied. In addition, the distribution of allelefrequencies in Pygmies is different from other populations, with Pygmiesshowing reduced polymorphism for the majority of cSNPs found.

TABLE 7A Bitter Taste Receptor Variants. Total SNPs/ LIST OF SEQ ID No.NONSY/ HAPLOTYPE NUMBER TOTAL NO: GENE No. Haplotypes¹ SEQUENCES (N =110)² FREQUENCY³ (NA/AA)⁴ T2R01 3/2/3 GC 79 0.718 47/48 GT 5 0.045 49/50AC 26 0.236 51/52 T2R03 3/1/2 C 109 0.990 53/54 T 1 0.010 55/56 T2R048/7/8 GATTCCG 8 0.073 57/58 GATTCCA 12 0.109 59/60 GATTCGG 50 0.45561/62 GATACCG 2 0.018 63/64 GACTCCA 34 0.309 65/66 GACTTCA 1 0.009 67/68GCCTCCA 1 0.009 69/70 AACTCCA 2 0.018 71/72 T2R05 7/6/7 GCCAGG 54 0.49173/74 GCCAAG 4 0.036 75/76 GCCGGG 5 0.045 77/78 GCTAGG 1 0.009 79/80TCCAGG 44 0.400 81/82 TCCAGT 1 0.009 83/84 TTTAGG 1 0.009 85/86 T2R076/6/5 TGCACG 98 0.891 87/88 (INCLUDING 1 TGCACA 2 0.018 89/90 STOP)TGCATG 3 0.027 91/92 TGTACA 6 0.055 93/94 CTCTCG 1 0.009 95/96 T2R086/5/6 CTATA 25 0.227 97/98 CTATG 80 0.727  99/100 CTACA 1 0.009 101/102CTGTG 2 0.018 103/104 CGATG 1 0.009 105/106 TTATG 1 0.009 107/108 T2R097/7/8 CCTTGGC 63 0.573 109/110 CCTTGGA 2 0.018 111/112 CCTTGTC 1 0.009113/114 CCTCGGC 35 0.318 115/116 CCTCAGC 1 0.009 117/118 CCATGGC 3 0.027119/120 CATTGGC 2 0.018 121/122 ACTTGGC 3 0.027 123/124 T2R10 6/3/4 TAT98 0.891 125/126 TAC 1 0.009 127/128 TCT 1 0.009 129/130 CAT 10 0.091131/132 T2R13 1/1/2 A 75 0.682 133/134 G 35 0.318 135/136 T2R14 4/2/3 AA107 0.973 137/138 AG 1 0.009 139/140 GA 2 0.018 141/142 T2R16 8/4/5 GCTA67 0.609 143/144 GCTG 28 0.255 145/146 GCGA 13 0.118 147/148 GTTA 10.009 149/150 ACTA 1 0.009 151/152 T2R38 5/5/7 CACCG 184 0.558 153/154(PTC) CATCA 1 0.003 155/156 GGCCA 1 0.003 157/158 GACCA 10 0.030 159/160GACCG 7 0.021 161/162 GACTA 1 0.003 163/164 GATCA 126 0.382 165/166T2R39 2/2/2 CA 107 0.973 167/168 TG 3 0.027 169/170 T2R40 3/3/5 CAG 900.818 171/172 CAA 8 0.073 173/174 CGG 1 0.009 175/176 AAG 9 0.082177/178 AGG 2 0.018 179/180 T2R41 4/2/3 CT 84 0.764 181/182 CA 1 0.009183/184 TT 25 0.227 185/186 T2R44 11/9/7  TAGCCTACG 27 0.245 187/188TAGGCTAGG 6 0.055 189/190 CAGCCTGCT 1 0.009 191/192 CAGCCCGCG 28 0.255193/194 CTGCCCGCG 45 0.409 195/196 CTGCTCGCG 1 0.009 197/198 CTACCCGCG 20.018 199/200 T2R46 6/5/6 TAACC 22 0.204 201/202 (INCLUDING 2 TAAGC 10.009 203/204 STOPS) TAGCC 6 0.056 205/206 TTGCC 75 0.694 207/208 TTGCT1 0.009 209/210 GAACC 3 0.028 211/212 T2R47 5/3/4 AAG 32 0.291 213/214AAT 76 0.691 215/216 AGG 1 0.009 217/218 GAG 1 0.009 219/220 T2R4812/9/9  GCAAATGCT 33 0.300 221/222 GCAAATGCC 53 0.482 223/224 GCAAATGTC3 0.027 225/226 GCAAATCCT 1 0.009 227/228 GCCAATGCC 5 0.045 229/230GAAAATGTC 1 0.009 231/232 ACAAATGCC 12 0.109 233/234 ACAAACGCC 1 0.009235/236 ACATGTGCC 1 0.009 237/238 T2R49 11/9/7  AGCCGATGA 37 0.378239/240 AGCCGATGG 2 0.020 241/242 AGCCAATGA 1 0.010 243/244 AGCAGATGA 30.031 245/246 AGAAGGCTA 19 0.194 247/248 AAAAGGCTA 13 0.133 249/250GGCCGATGA 23 0.235 251/252 T2R50 7/3/4 CGG 77 0.700 253/254 CGA 30 0.273255/256 CTG 1 0.009 257/258 TGA 2 0.018 259/260 T2R60 2/1/2 A 92 0.958261/262 T 4 0.042 263/264 127/95/109 (INCLUDING 3 STOP CODON)¹NONSY—Nonsynonymous Substitution ²N - Number examined. N does not applyto T2R38, T2R46, T2R49, T2R60. N of T2R38 = 330; T2R46 = 108; N of T2R49= 98; N of T2R60 = 96. ³Total Frequency = Total FRE of 5 Populations⁴SEQ ID NO: (NA/AA) - Sequence ID number for Nucleic Acid/Amino Acid.The reference, previously known sequence is indicated in bold;corresponding GenBank Accesion numbers are listed in Table 7C.

TABLE 7B Haplotype Distribution in Various Populations Number²/ SEQ IDLIST OF Total NO: GENE HAPLOTYPES CAM¹ AME¹ JAP¹ HUN¹ PYG¹ Frequency(NA/AA)³ T2R01 GC 39 6 12 14 8 79/0.718  47/48 GT 0 0 1 2 2 5/0.04549/50 AC 3 14 7 2 0 26/0.236  51/52 T2R03 C 41 20 20 18 10 109/0.990 53/54 T 1 0 0 0 0 1/0.010 55/56 T2R04 GATTCCG 7 0 0 0 1 8/0.073 57/58GATTCCA 2 9 1 0 0 12/0.109  59/60 GATTCGG 22 7 7 8 6 50/0.455  61/62GATACCG 2 0 0 0 0 2/0.018 63/64 GACTCCA 8 4 10 10 2 34/0.309  65/66GACTTCA 0 0 1 0 0 1/0.009 67/68 GCCTCCA 0 0 1 0 0 1/0.009 69/70 AACTCCA1 0 0 0 1 2/0.018 71/72 T2R05 GCCAGG 22 11 7 8 6 54/0.491  73/74 GCCAAG4 0 0 0 0 4/0.036 75/76 GCCGGG 4 0 0 0 1 5/0.045 77/78 GCTAGG 1 0 0 0 01/0.009 79/80 TCCAGG 9 9 13 10 3 44/0.400  81/82 TCCAGT 1 0 0 0 01/0.009 83/84 TTTAGG 1 0 0 0 0 1/0.009 85/86 T2R07 TGCACG 39 14 18 17 1098/0.891  87/88 TGCACA 0 0 1 1 0 2/0.018 89/90 TGCATG 3 0 0 0 0 3/0.02791/92 TGTACA 0 6 0 0 0 6/0.055 93/94 CTCTCG 0 0 1 0 0 1/0.009 95/96T2R08 CTATA 19 0 0 0 6 25/0.227  97/98 CTATG 20 20 19 17 4 80/0.727  99/100 CTACA 1 0 0 0 0 1/0.009 101/102 CTGTG 2 0 0 0 0 2/0.018 103/104CGATG 0 0 0 1 0 1/0.009 105/106 TTATG 0 0 1 0 0 1/0.009 107/108 T2R09CCTTGGC 27 16 5 10 5 63/0.573  109/110 CCTTGGA 2 0 0 0 0 2/0.018 111/112CCTTGTC 1 0 0 0 0 1/0.009 113/114 CCTCGGC 6 4 14 8 3 35/0.318  115/116CCTCAGC 0 0 1 0 0 1/0.009 117/118 CCATGGC 3 0 0 0 0 3/0.027 119/120CATTGGC 0 0 0 0 2 2/0.018 121/122 ACTTGGC 3 0 0 0 0 3/0.027 123/124T2R10 TAT 31 20 20 18 9 98/0.891  125/126 TAC 0 0 0 0 1 1/0.009 127/128TCT 1 0 0 0 0 1/0.009 129/130 CAT 10 0 0 0 0 10/0.091  131/132 T2R13 A40 10 5 10 10 75/0.682  133/134 G 2 10 15 8 0 35/0.318  135/136 T2R14 AA40 20 19 18 10 107/0.973  137/138 AG 0 0 1 0 0 1/0.009 139/140 GA 2 0 00 0 2/0.018 141/142 T2R16 GCTA 23 16 11 9 8 67/0.609  143/144 GCTG 6 4 99 0 28/0.255  145/146 GCGA 12 0 0 0 1 13/0.118  147/148 GTTA 0 0 0 0 11/0.009 149/150 ACTA 1 0 0 0 0 1/0.009 151/152 T2R38 N = 330 (PTC) CACCG184/0.558  153/154 CATCA 1/0.003 155/156 GGCCA 1/0.003 157/158 GACCA10/0.030  159/160 GACCG 7/0.021 161/162 GACTA 1/0.003 163/164 GATCA126/0.382  165/166 T2R39 CA 39 20 20 18 10 107/0.973  167/168 TG 3 0 0 00 3/0.027 169/170 T2R40 CAG 33 12 20 16 9 90/0.818  171/172 CAA 0 8 0 00 8/0.073 173/174 CGG 1 0 0 0 0 1/0.009 175/176 AAG 6 0 0 2 1 9/0.082177/178 AGG 2 0 0 0 0 2/0.018 179/180 T2R41 CT 41 13 11 9 10 84/0.764 181/182 CA 1 0 0 0 0 1/0.009 183/184 TT 0 7 9 9 0 25/0.227  185/186T2R44 TAGCCTACG 6 10 4 7 0 27/0.245  187/188 TAGGCTAGG 1 0 0 5 0 6/0.055189/190 CAGCCTGCT 1 0 0 0 0 1/0.009 191/192 CAGCCCGCG 0 10 14 4 028/0.255  193/194 CTGCCCGCG 32 0 2 2 9 45/0.409  195/196 CTGCTCGCG 0 0 00 1 1/0.009 197/198 CTACCCGCG 2 0 0 0 0 2/0.018 199/200 T2R46 N = 108TAACC 3 10 3 6 0 22/0.204  201/202 TAAGC 0 0 1 0 0 1/0.009 203/204 TAGCC1 0 0 5 0 6/0.056 205/206 TTGCC 34 10 16 5 10 75/0.694  207/208 TTGCT 10 0 0 0 1/0.009 209/210 GAACC 3 0 0 0 0 3/0.028 211/212 T2R47 AAG 7 10 411 0 32/0.291  213/214 AAT 34 10 16 6 10 76/0.691  215/216 AGG 1 0 0 0 01/0.009 217/218 GAG 0 0 0 1 0 1/0.009 219/220 T2R48 GCAAATGCT 7 10 4 120 33/0.300  221/222 GCAAATGCC 21 6 15 6 5 53/0.482  223/224 GCAAATGTC 00 0 0 3 3/0.027 225/226 GCAAATCCT 1 0 0 0 0 1/0.009 227/228 GCCAATGCC 04 1 0 0 5/0.045 229/230 GAAAATGTC 0 0 0 0 1 1/0.009 231/232 ACAAATGCC 110 0 0 1 12/0.109  233/234 ACAAACGCC 1 0 0 0 0 1/0.009 235/236 ACATGTGCC1 0 0 0 0 1/0.009 237/238 T2R49 N = 98 AGCCGATGA 23 0 0 6 8 37/0.378 239/240 AGCCGATGG 2 0 0 0 0 2/0.020 241/242 AGCCAATGA 1 0 0 0 0 1/0.010243/244 AGCAGATGA 3 0 0 0 0 3/0.031 245/246 AGAAGGCTA 1 6 9 3 019/0.194  247/248 AAAAGGCTA 1 4 7 1 0 13/0.133  249/250 GGCCGATGA 3 10 46 0 23/0.235  251/252 T2R50 CGG 40 10 4 13 10 77/0.700  253/254 CGA 1 1014 5 0 30/0.273  255/256 CTG 1 0 0 0 0 1/0.009 257/258 TGA 0 0 2 0 02/0.018 259/260 T2R60 N = 96 A 27 20 20 18 7 92/0.958  261/262 T 3 0 0 01 4/0.042 263/264 NOTES: ¹CAM = Cameroonian; AME = Amerindian; JAP =Japanese; HUN = Hungarian; PYG = Pygmy ²N - number examined (110 unlessotherwise indicated). Total Frequency = Total FRE of 5 Populations ⁴SEQID NO: (NA/AA) - Sequence ID number for Nucleic Acid/Amino Acid.

TABLE 7C Relationship of T2R Haplotypes to GenBank Reference SequenceGenBank Sequence SEQ ID Gene GenBank Accession GenBank AlleleNOs.(NA/AA)¹ NA NA Variants AA position AA Variants position T2R01AF227129 GC 47/48 332 G/A 111 Arg/His 616 C/T 206 Arg/Trp T2R03 AF227130C 53/54 349 C/T 117 Pro/Ser T2R04 AF227131 GATTCGG 61/62  8 G/A  3Arg/Gln  17 A/C  6 Tyr/Ser  20 T/C  7 Phe/Ser 186 T/A  62 Phe/Leu 221C/T  74 Thr/Met 268 C/G  96 Leu/Val 512 G/A 171 Ser/Asn T2R05 AF227132GCCAGG 73/74  77 G/T  26 Ser/Ile 235 C/T  79 Arg/Cys 338 C/T 113 Pro/Leu500 A/G 167 Tyr/Cys 638 G/A 213 Arg/Gln 881 G/T 294 Arg/Leu T2R07AF227133 TGCACG 87/88 254 T/C  85 Ile/Thr 538 G/T 180 Ala/Ser 640 C/T214 Arg/Stop 787 A/T 263 Thr/Ser 788 C/T 263 Thr/Met 912 G/A 304 Met/IleT2R08 AF227134 CTATA 97/98 142 C/T  48 Leu/Phe 370 T/G 124 Trp/Gly 496A/G 166 Arg/Gly 829 T/C 277 Tyr/His 922 A/G 308 Met/Val T2R09 AF227135CCTTGGC 109/110 201 C/A  67 Phe/Leu 381 C/A 127 Asn/Lys 450 T/A 150Asp/Glu 560 T/C 187 Val/Ala 697 G/A 233 Ala/Thr 867 G/T 289 Leu/Phe 880C/A 294 Leu/Met T2R10 AF227136 CAT 131/132 467 T/C 156 Met/Thr 521 A/C174 Lys/Thr 691 T/C 231 Ser/Pro T2R13 AF227137 A 133/134 776 A/G 259Asn/Ser T2R14 AF227138 AA 137/138 256 A/G  86 Thr/Ala 589 A/G 197Met/Val T2R16 AF227139 GCTG 145/146 301 G/A 101 Val/Met 481 C/T 161Pro/Ser 516 T/G 172 Asn/Lys 665 A/G 222 His/Arg T2R38 AF494231 GATCA165/166 145 C/G  49 Pro/Ala 239 A/G  80 His/Arg 785 C/T 262 Ala/Val 820C/T 274 Arg/Cys 886 G/A 296 Val/Ile T2R39 AF494230 CA 167/168 578 C/T193 Ser/Phe 589 A/G 197 Lys/Glu T2R40 AF494229 CAG 171/172 560 C/A 187Set/Tyr 817 A/G 273 Thr/Ala 871 G/A 291 Gly/Ser T2R41 AF494232 TT185/186 380 C/T 127 Pro/Leu 584 T/A 195 Val/Asp T2R44 AF494228 CAGCCCGCG193/194 103 T/C  35 Trp/Arg 484 A/T 162 Met/Leu 599 G/A 200 Cys/Tyr 649C/G 217 Gln/Glu 656 C/T 219 Pro/Leu 680 T/C 227 Val/Ala 718 A/G 240Ile/Val 827 C/G 276 Pro/Arg 843 G/T 281 Trp/Cys T2R46 AF494227 TTGCC207/208 106 T/G  36 Phe/Val 682 A/T 228 Met/Leu 749 A/G 250 Stop/Trp 834C/G 278 Ile/Met 862 C/T 288 Gln/Stop T2R47 AF494233 AAT 215/216 521 A/G174 His/Arg 577 A/G 193 Ile/Val 756 G/T 252 Leu/Phe T2R48 AF494234GCAAATGCC 223/224 94 G/A  32 Val/Ile 113 C/A  38 Thr/Lys 376 A/C 126Lys/Gln 456 A/T 152 Arg/Ser 673 A/G 225 Ile/Val 719 T/C 240 Ile/Thr 799G/C 267 Val/Leu 815 C/T 272 Pro/Leu 895 T/C 299 Cys/Arg T2R49 AF494236AGCCGATGA 239/240 235 A/G  79 Lys/Glu 421 G/A 141 Val/Ile 429 C/A 143His/Gln 442 C/A 148 His/Asn 516 G/A 172 Met/Ile 706 A/G 236 Ile/Val 755T/C 252 Phe/Ser 764 G/T 255 Arg/Leu 808 A/G 270 Ile/Val T2R50 AF494235CGA 255/256 155 C/T  52 Ala/Val 181 G/T  61 Ala/Ser 608 G/A 203 Cys/TyrT2R60 AY114094 A 261/262 595 A/T 199 Met/Leu ¹SEQ ID NO:(NA/AA)—Sequence ID number for Nucleic Acid/Amino Acid.

Of the cSNPs found, 92(72%) are nonsynonymous and 32(25%) aresynonymous. 42.5% of nonsynonymous substitutions are non-conservative,and amino acid changes were observed across the entire coding region ofthese genes. Interestingly, we observed two segregating pseudogenes(SPGs); that is, T2R genes for which both intact and nonsense versions(null alleles) were segregating in the human sample. One is the T2R46gene, which has two nonsense alleles, G749A and C862T. G749A has a nullallele frequency of ˜24% in all populations except Pygmy, while C862Twas observed only once, in Cameroonians. The other SPG was observed inT2R7 and although it was observed only in Amerindians, the null alleledisplayed a frequency of 30% in this population. Although only two SPGsseem to exist in the entire repertoire of T2R genes, our result isconsistent with observations in the olfactory receptor gene family, andsuggests that T2R SPGs exist at different frequencies in differentpopulations (Menashe et al., Hum. Mol. Genet. 11:1381-1390, 2002; Gilad,et al., Mol. Biol. Evol. 20:307-314, 2002).

Given the fact that different combinations of each polymorphic site in abitter receptor gene can explain the phenotypic variation for a bittercompound in humans (Kim, et al., Science 299:1221-1225, 2003), weinferred possible functional haplotypes with nonsynonymous substitutionsof each gene using Bayesian methods as implemented in the Phase package.In the 20 T2R genes that contain at least two cSNPs specifying aminoacid change, we identified 109 different haplotypes. The number ofhaplotypes ranged from 2 to 9 for each gene. With the exception of T2R44and T2R49, the most common haplotype of each gene was consistent acrosspopulations, and was observed in all five different populations.

To investigate the effects of natural selection on the bitter tastereceptor genes as a group, we performed neutrality tests using Tajima'sD statistic. Tajima's D compares the mean pairwise difference betweenrandomly chosen sequences in a sample with the number of segregatingsites (Tajima, Genetics 123:585-595, 1989). Tajima's D test is usuallyperformed under the assumption that population size has been constant.However, much evidence suggests that modern human populations are theproduct of an expansion that took place around 100,000 years ago, duringwhich the human population soared from an initial effective size ofroughly 10,000 (Excoffier, Curr. Opin. Genet. Dev. 12:675-682,2002;Klein, The Human Career: Human Biological and Cultural Origins, 1999;Harpending, et al., Annu. Rev. Genomics Hum. Genet. 1:361-385, 2000;Tishkoff, et al., Annu. Rev. Genomics Hum. Genet. 4:293-340,2003). Suchgrowth is important to consider in neutrality tests because it can bothmimic the effects of positive selection (i.e., selective sweeps) andobscure the effects of balancing selection (e.g. heterozygote advantage)(Bamshad, et al., Nat. Rev. Genet. 4:99-111, 2003). For this reason, wetested the hypothesis of selective neutrality under both the assumptionof constant population size and the assumption of 100-fold growth,100,000 years ago, as described by Wooding et al., (Am. J. Hum. Genet.74:637-646, 2004).

When considered individually, only two bitter taste genes, T2R16 andT2R49, showed significant departures from neutrality under both theassumption of constant population size and the assumption of populationgrowth (Table 8). T2R16 had a highly negative D-value (−2.152, P<0.01under growth) and T2R49 had a highly positive D-value (1.856, P<0.005under growth). One additional gene, T2R13, showed a significant positivedeparture from expectation only under the assumption of populationgrowth (p<0.025). Three more T2R4, T2R44, and T2R60 had D values thatwere high but not statistically significant under either model. Whenmultiple comparisons were taken into account using a Bonferronicorrection, none of the D values departed significantly fromexpectation, although under growth the D value for T2R49 failed to reachsignificance only marginally.

TABLE 8 Summary statistics for T2Rs Prob (Dsim > Dobs) No T2R# Seg. mpdπ (%) F_(ST) D Growth Growth 1 3 0.047 0.005 0.233 −1.618 0.995 0.912 33 0.545 0.057 0.103 −0.074 0.541 0.201 4 8 1.701 0.189 0.065 0.289 0.3010.126 5 7 0.973 0.109 0.067 −0.616 0.640 0.424 7 6 0.348 0.036 0.127−1.536 0.991 0.875 8 6 0.906 0.097 0.275 −0.451 0.649 0.349 9 7 0.6420.068 0.141 −1.193 0.915 0.717 10 6 0.568 0.061 0.136 −1.108 0.906 0.68913 1 0.438 0.048 0.341 1.473 0.095 0.010 14 4 0.544 0.057 0.469 −0.5520.635 0.349 16 8 0.152 0.017 0.150 −2.152 1.000 0.996 39 2 0.107 0.0110.021 −1.084 0.896 0.671 40 3 0.371 0.038 0.148 −0.614 0.690 0.429 41 40.745 0.081 0.231 −0.035 0.517 0.204 44 11 2.500 0.269 0.239 0.511 0.2330.078 46 6 0.975 0.105 0.117 −0.317 0.588 0.287 47 5 0.917 0.096 0.156−0.068 0.499 0.217 48 12 1.848 0.205 0.257 −0.493 0.676 0.354 49 113.590 0.386 0.327 1.856 0.031 0.005 50 7 1.269 0.141 0.246 −0.102 0.5310.221 60 2 0.515 0.054 0.236 0.540 0.243 0.076 Column headings are asfollows: T2R#, bitter taste receptor gene (e.g., 1 = T2R01); Seg.,Number of polymorphic nucleotide positions; mpd, mean pairwisedifference among randomly chosen sequences; pi, mean pairwise differenceper nucleotide; D, Tajima's D statistic calculated from Seg. and mpd (n= 110); Prob (Dsim > Dobs), probability that a D value simulated underthe given model exceeded the observed value. No growth indicates thatsimulations assumed a constant human population size. Growth indicatesthat simulations assumed that the human population size increased from10,000 to 1,000,000, 100,000 years ago.

Although none of the individual D values in our sample departedsignificantly from expectation, the distribution of D values did. Whenwe compared the mean D value observed in the 21 bitter taste receptorgenes with theoretical expectations under the assumptions of (i) nopopulation growth, and (ii) 100-fold growth, 100,000 years ago, the meanD value observed in our sample (−0.35) differed significantly fromexpectation under growth (−0.67) (P<0.01). Further, bootstrap resamplingtests showed that the mean observed D value was significantly greaterthan the mean D value (−1.00) in 160 genes resequenced as part of theNIH Environmental Genome Project (EGP). The EGP genes provide anappropriate comparison because they, like the taste receptors in oursample, are thought to be particularly important in mediatinginteractions between the human body and its environment (Wakefield,Eniviron. Health Perspect 110:A757-759, 2002). As shown in FIG. 3, theEGP genes showed a distribution of D values similar to that expectedunder the assumption of 100-fold growth, 100,000 years ago. Thus, notonly is the average D value in the bitter taste receptors significantlygreater than expected under reasonable assumptions about humanpopulation history (i.e., growth), it is significantly greater thanexpected given a large sample of comparable genes (i.e., environmentallyresponsive genes).

High values of Tajima's D are caused by a relative overabundance of SNPswith intermediate frequencies (i.e., frequencies near 50%). Such valuesoften indicate the presence of balancing natural selection; however,high D values can also be caused by population subdivision (Bamshad, etal., Nat. Rev. Genet. 4:99-111, 2003). To distinguish these alternativeswe analyzed the FST statistic, which takes values near zero in theabsence of population differentiation and values near unity in thepresence of extreme differentiation. FST values in our sample rangedfrom 0.02 to 0.47, with a mean of 0.25. Published FST values based onDNA sequence variation in humans are usually lower, often falling around0.15 (Tishkoff et al., Annu. Rev. Genomics Hum. Genet. 4:293-340, 2003).For example, in a study of 25,549 SNPs, Akey et al. found an average FSTof 0.123 (Genome Res. 12:1805-1814,2002). Bootstrap tests showed thatthe mean value observed in our dataset is significantly higher than thatin the TSC dataset. The preponderance of high FST values in our samplesuggests that human populations differ more with respect to variation inthe bitter taste receptor genes than they do with respect to most otherregions of the genome.

The patterns of genetic variation found in the bitter taste receptorgenes in general are illustrated by the T2R49 gene. Among the genes weexamined, T2R49 had the highest value of Tajima's D (1.86) and the thirdhighest FST (0.33). The value of Tajima's D in T2R49 is significantlygreater than expected (P<0.05 under constant population size, P<0.01under growth). The FST value is significantly greater than zero (P<0.01)and exceeds more than 85% of the FST values reported by Akey et al.(Genome Res. 12:1805-1814, 2002). A minimum spanning tree relating T2R49haplotypes showed that two common, distinct haplotype clusters differingfrom each other by six nucleotide substitutions (including four aminoacid substitutions) were present. The presence of two common, butdistinct, clusters causes a high value of Tajima's D at this locus. Asshown in FIG. 4, each cluster was common in a different geographicalregion. While cluster 1 accounted for 91.5% of observations in Africaand Europe, cluster 2 accounted for 65.0% of observations in Asia andAmerindians. The high frequency of each cluster in a differentgeographical region causes a high FST value at this locus. Further, nineout of 11 nucleotide substitutions in T2R49 cause amino acidsubstitutions. This ratio is extremely high compared to that observed inmost genes, and is consistent with the hypothesis that positive naturalselection has been active in the region (McDonald, et al., Nature351:652-654, 1991; Nekrutenko, et al., Genome Res. 12:198-202, 2002).

Example 5 Detecting Single Nucleotide Alterations

T2R bitter taste receptor single nucleotide alterations, whethercategorized as SNPs or new mutations can be detected by a variety oftechniques in addition to merely sequencing the target sequence.Constitutional single nucleotide alterations can arise either from newgermline mutations, or can be inherited from a parent who possesses aSNP or mutation in their own germline DNA. The techniques used inevaluating either somatic or germline single nucleotide alterationsinclude hybridization using allele specific oligonucleotides (ASOs)(Wallace et al., CSHL Symp. Quant. Biol. 51:257-261, 1986; Stoneking etal., Am. J. Hum. Genet. 48:370-382, 1991), direct DNA sequencing (Churchand Gilbert, Proc. Natl. Acad. Sci USA 81:1991-1995, 1988), the use ofrestriction enzymes (Flavell et al., Cell 15:25, 1978; Geever et al.,1981), discrimination on the basis of electrophoretic mobility in gelswith denaturing reagent (Myers and Maniatis, Cold Spring Harbor Symp.Quant. Biol. 51:275-284, 1986), RNase protection (Myers et al., Science230:1242, 1985), chemical cleavage (Cotton et al., Proc. Natl. Acad.Sci. USA 85:4397-4401, 1985), and the ligase-mediated detectionprocedure (Landegren et al., Science 241:1077, 1988).

Allele-specific oligonucleotide hybridization (ASOH) involveshybridization of probes to the sequence, stringent washing, and signaldetection. Other new methods include techniques that incorporate morerobust scoring of hybridization. Examples of these procedures includethe ligation chain reaction (ASOH plus selective ligation andamplification), as disclosed in Wu and Wallace (Genomics 4:560-569,1989); mini-sequencing (ASOH plus a single base extension) as discussedin Syvanen (Meth. Mol. Biol. 98:291-298, 1998); and the use of DNA chips(miniaturized ASOH with multiple oligonucleotide arrays) as disclosed inLipshutz et al. (BioTechniques 19:442-447, 1995). Alternatively, ASOHwith single- or dual-labeled probes can be merged with PCR, as in the5′-exonuclease assay (Heid et al., Genome Res. 6:986-994, 1996), or withmolecular beacons (as in Tyagi and Kramer, Nat. Biotechnol. 14:303-308,1996).

Another technique is dynamic allele-specific hybridization (DASH), whichinvolves dynamic heating and coincident monitoring of DNA denaturation,as disclosed by Howell et al. (Nat. Biotech. 17:87-88, 1999). A targetsequence is amplified by PCR in which one primer is biotinylated. Thebiotinylated product strand is bound to a streptavidin-coated microtiterplate well, and the non-biotinylated strand is rinsed away with alkaliwash solution. An oligonucleotide probe, specific for one allele, ishybridized to the target at low temperature. This probe forms a duplexDNA region that interacts with a double strand-specific intercalatingdye. When subsequently excited, the dye emits fluorescence proportionalto the amount of double-stranded DNA (probe-target duplex) present. Thesample is then steadily heated while fluorescence is continuallymonitored. A rapid fall in fluorescence indicates the denaturingtemperature of the probe-target duplex. Using this technique, asingle-base mismatch between the probe and target results in asignificant lowering of melting temperature (T_(m)) that can be readilydetected.

Oligonucleotides specific to normal or allelic sequences can bechemically synthesized using commercially available machines. Theseoligonucleotides can then be labeled radioactively with isotopes (suchas ³²P) or non-radioactively, with tags such as biotin (Ward and Langeret al., Proc. Natl. Acad. Sci. USA 78:6633-6657, 1981), and hybridizedto individual DNA samples immobilized on membranes or other solidsupports by dot-blot or transfer from gels after electrophoresis. Thesespecific sequences are visualized by methods such as autoradiography orfluorometric (Landegren et al., Science 242:229-237, 1989) orcoloriinetric reactions (Gebeyehu et al., Nucleic Acids Res.15:4513-4534, 1987). Using an ASO specific for a normal allele, theabsence of hybridization would indicate a mutation in the particularregion of the gene, or a deleted gene. In contrast, if an ASO specificfor a mutant allele hybridizes to a sample then that would indicate thepresence of a mutation in the region defined by the ASO.

A variety of other techniques can be used to detect the mutations orother variations in DNA. Merely by way of example, see U.S. Pat. Nos.4,666,828; 4,801,531; 5,110,920; 5,268,267; 5,387,506; 5,691,153;5,698,339; 5,736,330; 5,834,200; 5,922,542; and 5,998,137 for suchmethods. Additional methods include fluorescence polarization methodssuch as those developed by Pui Kwok and colleagues (see, e.g., Kwok,Hum. Mutat., 19(4):315-23, 2002), microbead methods such as thosedeveloped by Mark Chee at Illumina (see, e.g., Oliphant et al.,Biotechniques. 2002 June; Suppl:56-8, 60-61, Shen et al., Genet. Eng.News, 23(6), 2003), and mass spectrophotometery methods such as thosebeing developed at Sequenom (on the Web at sequenom.com) (see, e.g.Jurinke et al., Methods Mol Biol. 187:179-92, 2002; Amexis et al., ProcNatl Acad Sci USA 98(21):12097-102, 2001; Jurinke et al., Adv BiochemEng Biotechnol. 2002; 77:57-74; Storm et al., Meth. Mol. Biol., 212:241262, 2002; Rodi et al., BioTechniques., 32:S62 S69, 2002); U.S. Pat. No.6,300,076; and WO9820166).

Example 6 Differentiation of Individuals Homozygous versus Heterozygousfor Mutation(s)

Since it is believed that the haplotype of any taste receptor caninfluence the perception of taste by a subject, it may sometimes bebeneficial to determine whether a subject is homozygous or heterozygousfor SNPs within any one or more of the T2R bitter taste receptorsdescribed herein.

By way of example, the oligonucleotide ligation assay (OLA), asdescribed at Nickerson et al. (Proc. Natl. Acad. Sci. USA 87:8923-8927,1990), allows the differentiation between individuals who are homozygousversus heterozygous for alleles or SNPs indicated in FIG. 1 or Table 7.This feature allows one to rapidly and easily determine whether anindividual is homozygous for at least one taste receptor variant, whichcondition believed to influence taste perception, particularly bittertaste reception, in the individual. Alternatively, OLA can be used todetermine whether a subject is homozygous for either of these mutations.

As an example of the OLA assay, when carried out in microtiter plates,one well is used for the determination of the presence of the T2R bittertaste allele in the T2R1 gene that contains an A at nucleotide position332 (numbering from SEQ ID NO: 1) and a second well is used for thedetermination of the presence of the T2R bitter taste allele in the samegene that contains a G at that nucleotide position in the alternateallele sequence. Thus, the results for an individual who is heterozygousfor the mutation will show a signal in each of the A and G wells.

Example 7 Bitter Taste Profiles

With the provision herein of specific SNPs within the family of bittertaste receptors that are linked to bitter taste sensitivity to one ormore bitter compounds, as well as SNPs and haplotypes that can be usedto distinguish populations from each other, genetic profiles thatprovide information on the bitter taste perception and/or regionality ofa subject are now enabled. Such profiles are useful in myriadapplications, including for instance selecting subjects for inclusion in(or exclusion from) a protocol (such as a taste test),

Bitter taste-related genetic profiles comprise the distinct andidentifiable pattern of alleles or haplotypes, or sets of alleles orhaplotypes, of the SNPs in bitter taste receptor molecules identifiedherein. The set of bitter taste receptors analyzed in a particularprofile will usually include at least one of the following: T2R1, T2R3,T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R38, T2R39,T2R40, T2R41, T2R43, T2R44, T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, orT2R60.

By way of example, any subset of the molecules listed in FIG. 1 or Table7 (or corresponding to the molecules in these lists) may be included ina single bitter taste profile. Specific examples of such subsets includethose molecules that show a SNP that introduces a stop codon (e.g., thevariant of T2R44 at position 843; the variant of T2R46 at position 749or 86, or the variant of T2R48 at position 885); that show a novel SNP(e.g., those T2R genes with a “new” SNP indicated in FIG. 1); and soforth. Alternatively, gene profiles may be further broken down by thetype of bitter taste receptors included in the profile, for instance,those which all occur on a single chromosome (e.g., CH 5, 7, or 12), orall of the haplotypes/isoforms of a single T2R gene. Specificcontemplated subsets of sequences will include at least one of thefollowing: two or more nucleotides selected from SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, and 263; or two or more polypeptides having asequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,and 264; or at least one fragment from each of two or more of suchmolecules, which fragment overlaps a variant defined in any one of SEQID NOs: 1-46.

The alleles/haplotypes of each bitter taste receptor included in aspecific profile can be determined in any of various known ways,including specific methods provided herein. One particular contemplatedmethod for detecting and determining the genotype and/or haplotype ofmultiple bitter taste receptors employs an array of allele-specificoligonucleotides which are used for qualitative and/or quantitativehybridization detection of the presence of specific alleles or SNPs in asample from a subject.

Optionally, a subject's bitter taste profile can be correlated with oneor more appropriate inhibitors or blockers of bitter taste, or othercompounds that influence the ability of a subject to perceive a taste,which may be correlated with a control (or set of control) profile(s)condition linked to or associated with, for instance, sensitivity to oneor a set of bitter compounds. Optionally, the subject's bitter tasteprofile can be correlated with one or more appropriate treatments, forinstance, treatments with compounds that inhibit or enhance the activityof one or more of the bitter taste alleles identified in the profile, orcompositions in which the bitter taste of a component is specificallymasked by a blocker that is added based on the information in theprofile.

Example 8 Expression of T2R Bitter Taste Receptor Variant Polypeptides

The expression and purification of proteins, such as a T2R bitter tastereceptor variant protein, can be performed using standard laboratorytechniques, though these techniques are preferentially adapted to befitted to express the T2R proteins. By way of example, techniques forexpression of T2R family proteins is discussed in Wu et al., PNAS99:2392-2397, 2002 (incorporated herein by reference in its entirety).

Additional examples of such method adaptations are discussed orreferenced herein. After expression, purified protein may be used forfunctional analyses, antibody production, diagnostics, and patienttherapy. Furthermore, the DNA sequences of the T2R bitter taste receptorvariant cDNAs can be manipulated in studies to understand the expressionof the gene and the function of its product. Variant or allelic forms ofa human T2R bitter taste receptor genes may be isolated based uponinformation contained herein, and may be studied in order to detectalteration in expression patterns in terms of relative quantities,tissue specificity and functional properties of the encoded T2R bittertaste receptor variant protein (e.g., influence on perception of taste).Partial or full-length cDNA sequences, which encode for the subjectprotein, may be ligated into bacterial expression vectors. Methods forexpressing large amounts of protein from a cloned gene introduced intoEschlerchia coli (E. coli) or more preferably baculovirus/Sf9 cells maybe utilized for the purification, localization and functional analysisof proteins. For example, fusion proteins consisting of amino terminalpeptides encoded by a portion of a gene native to the cell in which theprotein is expressed (e.g., a E. coli lacZ or trpE gene for bacterialexpression) linked to a T2R bitter taste receptor variant protein may beused to prepare polyclonal and monoclonal antibodies against theseproteins. Thereafter, these antibodies may be used to purify proteins byimmunoaffinity chromatography, in diagnostic assays to quantitate thelevels of protein and to localize proteins in tissues and individualcells by immunofluorescence.

Intact native protein may also be produced in large amounts forfunctional studies. Methods and plasmid vectors for producing fusionproteins and intact native proteins in culture are well known in theart, and specific methods are described in Sambrook et al (In MolecularCloning: A Laboratory Manual, Ch 17, CSHL, New York, 1989). Such fusionproteins may be made in large amounts, are easy to purify, and can beused to elicit antibody response. Native proteins can be produced inbacteria by placing a strong, regulated promoter and an efficientribosome-binding site upstream of the cloned gene. If low levels ofprotein are produced, additional steps may be taken to increase proteinproduction; if high levels of protein are produced, purification isrelatively easy. Suitable methods are presented in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and arewell known in the art. Often, proteins expressed at high levels arefound in insoluble inclusion bodies. Methods for extracting proteinsfrom these aggregates are described by Sambrook et al. (In MolecularCloning: A Laboratory Manual, Ch. 17, CSHL, New York, 1989). Vectorsystems suitable for the expression of lacZ fusion genes include the pURseries of vectors (Ruther and Muller-Hill, EMBO J. 2; 1791, 1983),pEX1-3 (Stanley and Luzio, EMBO J. 3:1429, 1984) and pMR100 (Gray etal., Proc. Natl. Acad. Sci. USA 79:6598, 1982). Vectors suitable for theproduction of intact native proteins include pKC30 (Shimatake andRosenberg, Nature 292:128, 1981), pKK177-3 (Amann and Brosius, Gene40:183, 1985) and pET-3 (Studiar and Moffatt, J. Mol. Biol. 189:113,1986).

Fusion proteins may be isolated from protein gels, lyophilized, groundinto a powder and used as an antigen. The DNA sequence can also betransferred from its existing context to other cloning vehicles, such asother plasmids, bacteriophages, cosmids, animal viruses and yeastartificial chromosomes (YACs) (Burke et al., Science 236:806-812, 1987).These vectors may then be introduced into a variety of hosts includingsomatic cells, and simple or complex organisms, such as bacteria, fungi(Timberlake and Marshall, Science 244:1313-1317, 1989), invertebrates,plants (Gasser and Fraley, Science 244:1293, 1989), and animals (Purselet al., Science 244:1281-1288, 1989), which cell or organisms arerendered transgenic by the introduction of the heterologous cDNA.

For expression in mammalian cells, the cDNA sequence may be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci USA 78:2072-2076,1981), and introduced into cells, such as monkey COS-1 cells (Gluzman,Cell 23:175-182, 1981), to achieve transient or long-term expression.The stable integration of the chimeric gene construct may be maintainedin mammalian cells by biochemical selection, such as neomycin (Southernand Berg, J. Mol. Appl. Genet. 1:327-341, 1982) and mycophenolic acid(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR or other in vitroamplification.

The cDNA sequence (or portions derived from it) or a mini gene (a cDNAwith an intron and its own promoter) may be introduced into eukaryoticexpression vectors by conventional techniques. These vectors aredesigned to permit the transcription of the cDNA in eukaryotic cells byproviding regulatory sequences that initiate and enhance thetranscription of the cDNA and ensure its proper splicing andpolyadenylation. Vectors containing the promoter and enhancer regions ofthe SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus andpolyadenylation and splicing signal from SV40 are readily available(Mulligan et al., Proc. Natl. Acad. Sci. USA 78:1078-2076, 1981; Gormanet al., Proc. Natl. Acad. Sci USA 78:6777-6781, 1982). The level ofexpression of the cDNA can be manipulated with this type of vector,either by using promoters that have different activities (for example,the baculovirus pAC373 can express cDNAs at high levels in S. frugiperdacells (Summers and Smith, In Genetically Altered Viruses and theEnvironment, Fields et al. (Eds.) 22:319-328, CSHL Press, Cold SpringHarbor, N.Y., 1985) or by using vectors that contain promoters amenableto modulation, for example, the glucocorticoid-responsive promoter fromthe mouse mammary tumor virus (Lee et al., Nature 294:228, 1982). Theexpression of the cDNA can be monitored in the recipient cells 24 to 72hours after introduction (transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell Biol 1:486, 1981) or Epstein-Barr(Sugden et al., Mol. Cell Biol. 5:410, 1985). Alternatively, one canalso produce cell lines that have integrated the vector into genomicDNA. Both of these types of cell lines produce the gene product on acontinuous basis. One can also produce cell lines that have amplifiedthe number of copies of the vector (and therefore of the cDNA as well)to create cell lines that can produce high levels of the gene product(Alt et al., J. Biol. Chem. 253:1357, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is now a conventional technique. The vectors areintroduced into the recipient cells as pure DNA (transfection) by, forexample, precipitation with calcium phosphate (Graham and vander Eb,Virology 52:466, 1973) or strontium phosphate (Brash et al., Mol. CellBiol. 7:2013, 1987), electroporation (Neumann et al., EMBO J 1:841,1982), lipofection (Felgner et al., Proc. Natl. Acad. Sci USA 84:7413,1987), DEAE dextran (McCuthan et al., J Natl. Cancer Inst. 41:351,1968), microinjection (Mueller et al., Cell 15:579, 1978), protoplastfusion (Schafner, Proc. Natl. Acad Sci. USA 77:2163-2167, 1980), orpellet guns (Klein et al., Nature 327:70, 1987). Alternatively, thecDNA, or fragments thereof, can be introduced by infection with virusvectors. Systems are developed that use, for example, retroviruses(Bernstein et al., Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al.,J. Virol. 57:267, 1986), or Herpes virus (Spaete et al., Cell 30:295,1982). T2R encoding sequences can also be delivered to target cells invitro via non-infectious systems, for instance liposomes.

These eukaryotic expression systems can be used for studies of T2Rbitter taste receptor variant encoding nucleic acids and mutant forms ofthese molecules, T2R bitter taste receptor variant proteins and mutantforms of these proteins. The eukaryotic expression systems may also beused to study the function of the normal complete protein, specificportions of the protein, or of naturally occurring or artificiallyproduced mutant proteins.

Using the above techniques, the expression vectors containing a T2R genesequence or cDNA, or fragments or variants or mutants thereof, can beintroduced into human cells, mammalian cells from other species ornon-mammalian cells as desired. The choice of cell is determined by thepurpose of the treatment. For example, monkey COS cells (Gluzman, Cell23:175-182, 1981) that produce high levels of the SV40 T antigen andpermit the replication of vectors containing the SV40 origin ofreplication may be used. Similarly, Chinese hamster ovary (CHO), mouseNIH 3T3 fibroblasts or human fibroblasts or lymphoblasts may be used.

The present disclosure thus encompasses recombinant vectors thatcomprise all or part of the T2R bitter taste receptor variant gene orcDNA sequences, for expression in a suitable host. The T2R bitter tastereceptor DNA is operatively linked in the vector to an expressioncontrol sequence in the recombinant DNA molecule so that a T2R bittertaste receptor polypeptide can be expressed. The expression controlsequence may be selected from the group consisting of sequences thatcontrol the expression of genes of prokaryotic or eukaryotic cells andtheir viruses and combinations thereof. The expression control sequencemay be specifically selected from the group consisting of the lacsystem, the trp system, the tac system, the trc system, major operatorand promoter regions of phage lambda, the control region of fd coatprotein, the early and late promoters of SV40, promoters derived frompolyoma, adenovirus, retrovirus, baculovirus and simian virus, thepromoter for 3-phosphoglycerate kinase, the promoters of yeast acidphosphatase, the promoter of the yeast alpha-mating factors andcombinations thereof.

One highly successful method of expressing T2R's to date is to engineeran amino-terminal portion of rhodopsin (e.g., the first 26 amino acidsthereof) onto the amino terminal end and express the resultant fusionprotein, for instance in a baculovirus/Sf9 cell system. By way ofexample, methods for expressing T2Rs in vitro are described inChandrashekar et aL (Cell 100:703-711, 2000), which is incorporatedherein by reference in its entirety. See also Vince et al., PNAS99:2392-2397, 2002.

The host cell, which may be transfected with the vector of thisdisclosure, may be selected from the group consisting of E. coli,Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or otherbacilli; other bacteria; yeast; fungi; insect; mouse or other animal; orplant hosts; or human tissue cells.

It is appreciated that for mutant or variant T2R bitter taste receptorDNA sequences, similar systems are employed to express and produce themutant product. In addition, fragments of a T2R bitter taste receptorprotein can be expressed essentially as detailed above. Such fragmentsinclude individual T2R bitter taste receptor protein domains orsub-domains, as well as shorter fragments such as peptides. T2R bittertaste receptor protein fragments having therapeutic properties may beexpressed in this manner also, including for instance substantiallysoluble fragments.

Example 9 Production of Protein Specific Binding Agents

Monoclonal or polyclonal antibodies may be produced to either a wildtypeor reference T2R bitter taste receptor protein or specific allelic formsof these proteins, for instance particular portions that contain adifferential amino acid encoded by a SNP and therefore may provide adistinguishing epitope. Optimally, antibodies raised against theseproteins or peptides would specifically detect the protein or peptidewith which the antibodies are generated. That is, an antibody generatedto the specified bitter taste receptor protein or a fragment thereofwould recognize and bind that protein and would not substantiallyrecognize or bind to other proteins found in human cells. In someembodiments, an antibody is specific for (or measurably preferentiallybinds to) an epitope in a variant protein (e.g., an allele of a T2Rbitter taste receptor as described herein) versus the reference protein,or vice versa, as discussed more fully herein.

The determination that an antibody specifically detects a target proteinor form of the target protein is made by any one of a number of standardimmunoassay methods; for instance, the western blotting technique,(Sambrook et al., In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989). To determine that a given antibody preparation (such as oneproduced in a mouse) specifically detects the target protein by westernblotting, total cellular protein is extracted from human cells (forexample, lymphocytes) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are then transferred to amembrane (for example, nitrocellulose) by western blotting, and theantibody preparation is incubated with the membrane. After washing themembrane to remove non-specifically bound antibodies, the presence ofspecifically bound antibodies is detected by the use of an anti-mouseantibody conjugated to an enzyme such as alkaline phosphatase.Application of an alkaline phosphatase substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immunolocalized alkalinephosphatase. Antibodies that specifically detect the target proteinwill, by this technique, be shown to bind to the target protein band(which will be localized at a given position on the gel determined byits molecular weight). Non-specific binding of the antibody to otherproteins may occur and may be detectable as a weak signal on the Westernblot. The non-specific nature of this binding will be recognized by oneskilled in the art by the weak signal obtained on the Western blotrelative to the strong primary signal arising from the specificantibody-target protein binding.

Substantially pure T2R bitter taste receptor protein or protein fragment(peptide) suitable for use as an immunogen may be isolated from thetransfected or transformed cells as described above. Concentration ofprotein or peptide in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms per milliliter. Monoclonal or polyclonal antibody to theprotein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of the target protein identified andisolated as described can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature 256:495-497,1975) or derivative methods thereof. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein over a periodof a few weeks. The mouse is then sacrificed, and the antibody-producingcells of the spleen isolated. The spleen cells are fused by means ofpolyethylene glycol with mouse myeloma cells, and the excess un-fusedcells destroyed by growth of the system on selective media comprisingaminopterin (HAT media). The successfully fused cells are diluted andaliquots of the dilution placed in wells of a microtiter plate wheregrowth of the culture is continued. Antibody-producing clones areidentified by detection of antibody in the supernatant fluid of thewells by immunoassay procedures, such as ELISA, as originally describedby Engvall (Meth. Enzymol. 70:419-439, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use. Detailed procedures for monoclonalantibody production are described in Harlow and Lane (Antibodies, ALaboratory Manual, CSHL, New York, 1988).

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofa single protein can be prepared by immunizing suitable animals with theexpressed protein, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related both to the antigen and the host species. Forexample, small molecules tend to be less immunogenic than others and mayrequire the use of carriers and adjuvant. Also, host animals vary inresponse to site of inoculations and dose, with either inadequate orexcessive doses of antigen resulting in low titer antisera. Small doses(ng level) of antigen administered at multiple intradermal sites appearto be most reliable. An effective immunization protocol for rabbits canbe found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-991,1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al. (In Handbook of Experimental Immunology,Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration ofantibody is usually in the range of about 0.1 to 0.2 mg/ml of serum(about 12 μM). Affinity of the antisera for the antigen is determined bypreparing competitive binding curves, as described, for example, byFisher (Manual of Clinical Immunology, Ch. 42, 1980).

C. Antibodies Raised Against Synthetic Peptides

A third approach to raising antibodies against a specific T2R bittertaste receptor protein or peptide (e.g., a peptide that is specific to avariant T2R bitter taste receptor such as those disclosed herein) is touse one or more synthetic peptides synthesized on a commerciallyavailable peptide synthesizer based upon the predicted amino acidsequence of the protein or peptide. Polyclonal antibodies can begenerated by injecting these peptides into, for instance, rabbits ormice.

D. Antibodies Raised by Injection of Encoding Sequence

Antibodies may be raised against proteins and peptides by subcutaneousinjection of a DNA vector that expresses the desired protein or peptide,or a fragment thereof, into laboratory animals, such as mice. Deliveryof the recombinant vector into the animals may be achieved using ahand-held form of the Biolistic system (Sanford et al., Particulate Sci.Technol. 5:27-37, 1987) as described Tang et al. (Nature 356:152-154,1992). Expression vectors suitable for this purpose may include thosethat express the T2R bitter taste receptor-encoding sequence under thetranscriptional control of either the human β-actin promoter or thecytomegalovirus (CMV) promoter.

Antibody preparations prepared according to these protocols are usefulin quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample; or for immunolocalization of the specifiedprotein.

Optionally, antibodies, e.g., bitter taste receptor-specific monoclonalantibodies, can be humanized by methods known in the art. Antibodieswith a desired binding specificity can be commercially humanized(Scotgene, Scotland, UK; Oxford Molecular, Palo Alto, Calif.).

E. Antibodies Specific for Specific T2R Taste Receptor Variants

With the provision of several variant T2R bitter taste receptorproteins, the production of antibodies that specifically recognize theseprotein variants (and peptides derived therefrom) is enabled. Inparticular, production of antibodies (and fragments and engineeredversions thereof) that recognize at least one variant receptor with ahigher affinity than they recognize a corresponding wild type T2R bittertaste receptor, or another bitter taste receptor, is beneficial, as theresultant antibodies can be used in analysis, diagnosis and treatment(e.g., inhibition or enhancement of bitter taste perception), as well asin study and examination of the T2R bitter taste receptor proteinsthemselves.

In particular embodiments, it is beneficial to generate antibodies froma peptide taken from a variation-specific region of the desired T2Rbitter taste receptor protein. By way of example, such regions includeany peptide (usually four or more amino acids in length) that overlapswith one or more of the SNP-encoded variants described herein. Moreparticularly, it is beneficial to raise antibodies against peptides offour or more contiguous amino acids that overlap the variants identifiedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 40, 42, or 46, and particularly which comprise at least fourcontiguous amino acids including the residue(s) indicated in FIG. 1 orTable 7 to be variable in different alleles of the specified T2Rputative bitter taste receptors/isoforms.

Longer peptides also can be used, and in some instances will produce astronger or more reliable immunogenic response. Thus, it is contemplatedin some embodiments that more than four amino acids are used to elicitthe immune response, for instance, at least 5, at least 6, at least 8,at least 10, at least 12, at least 15, at least 18, at least 20, atleast 25, or more, such as 30, 40, 50, or even longer peptides. Also, itwill be understood by those of ordinary skill that it is beneficial insome instances to include adjuvants and other immune response enhancers,including passenger peptides or proteins, when using peptides to inducean immune response for production of antibodies.

Embodiments are not limited to antibodies that recognize epitopescontaining the actual mutation identified in each variant. Instead, itis contemplated that variant-specific antibodies also may each recognizean epitope located anywhere throughout the specified T2R bitter tastereceptor variant molecule, which epitopes are changed in conformationand/or availability because of the activating mutation. Antibodiesdirected to any of these variant-specific epitopes are also encompassedherein.

By way of example, the following references provide descriptions ofmethods for making antibodies specific to mutant proteins: Hills et al.,(Int. J. Cancer, 63: 537-543, 1995); Reiter & Maihle (Nucleic AcidsRes., 24: 4050-4056, 1996); Okamoto et al. (Br. J. Cancer, 73:1366-1372, 1996); Nakayashiki et al., (Jpn. J. Cancer Res., 91:1035-1043, 2000); Gannon et al. (EMBO J., 9: 1595-1602, 1990); Wong etal. (Cancer Res., 46: 6029-6033, 1986); and Carney et al. (J. CellBiochem., 32: 207-214, 1986). Similar methods can be employed togenerate antibodies specific to specific T2R bitter taste receptorvariants.

Example 10 Knockout and Overexpression Transgenic Animals

Mutant organisms that under-express or over-express one or more specificalleles (isoforms) of one or more specific bitter taste receptor proteinare useful for research. Such mutants allow insight into thephysiological and/or psychological role of bitter taste perception in ahealthy and/or pathological organism. These “mutants” are “geneticallyengineered,” meaning that information in the form of nucleotides hasbeen transferred into the mutant's genome at a location, or in acombination, in which it would not normally exist. Nucleotidestransferred in this way are said to be “non-native.” For example, anon-bitter taste receptor promoter inserted upstream of a native bittertaste receptor-encoding sequence would be non-native. An extra copy of aspecific bitter taste receptor gene on a plasmid, transformed into acell, would be non-native.

Mutants may be, for example, produced from mammals, such as mice orrats, that either express, over-express, or under-express a specificallelic variant or haplotype or diplotype of a defined bitter tastereceptor (or combination of bitter taste receptors), or that do notexpress a specified receptor (or combination of receptors) at all.Over-expression mutants are made by increasing the number of specifiedgenes in the organism, or by introducing a specific taste receptorallele into the organism under the control of a constitutive orinducible or viral promoter such as the mouse mammary tumor virus (MMTV)promoter or the whey acidic protein (WAP) promoter or themetallothionein promoter. Mutants that under-express a taste receptor,or that do not express a specific allelic variant of a taste receptor,may be made by using an inducible or repressible promoter, or bydeleting the taste receptor gene, or by destroying or limiting thefunction of the taste receptor gene, for instance by disrupting the geneby transposon insertion.

Antisense genes or molecules (such as siRNAs) may be engineered into theorganism, under a constitutive or inducible promoter, to decrease orprevent expression of a specific T2R bitter taste receptor, as known tothose of ordinary skill in the art.

A mutant mouse over-expressing a heterologous protein (such as a variantT2R bitter taste receptor protein) may be made by constructing a plasmidhaving a bitter taste receptor allele encoding sequence driven by apromoter, such as the mouse mammary tumor virus (MMTV) promoter or thewhey acidic protein (WAP) promoter. This plasmid may be introduced intomouse oocytes by microinjection. The oocytes are implanted intopseudopregnant females, and the litters are assayed for insertion of thetransgene. Multiple strains containing the transgene are then availablefor study.

WAP is quite specific for mammary gland expression during lactation, andMMTV is expressed in a variety of tissues including mammary gland,salivary gland and lymphoid tissues. Many other promoters might be usedto achieve various patterns of expression, e.g., the metallothioneinpromoter.

An inducible system may be created in which the subject expressionconstruct is driven by a promoter regulated by an agent that can be fedto the mouse, such as tetracycline. Such techniques are well known inthe art.

A mutant knockout animal (e.g., mouse) from which a specific tastereceptor gene is deleted can be made by removing all or some of thecoding regions of the gene from embryonic stem cells. The methods ofcreating deletion mutations by using a targeting vector have beendescribed (Thomas and Capecchi, Cell 51:503-512, 1987).

Example 11 Knock-In Organisms

In addition to knock-out systems, it is also beneficial to generate“knock-ins” that have lost expression of the native protein but havegained expression of a different, usually mutant or identified allelicform of the same protein. By way of example, any one or more of theallelic protein isoforms provided herein (e.g., as shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 40, 42,and 46, or in SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, and 264) can be expressedin a knockout background in order to provide model systems for studyingthe effects of these mutants. In particular embodiments, the resultantknock-in organisms provide systems for studying taste reception, forinstance how the taste of specific molecules is perceived.

Those of ordinary skill in the relevant art know methods of producingknock-in organisms. See, for instance, Rane et al. (Mol. Cell Biol., 22:644-656, 2002); Sotillo et al. (EMBO J., 20: 6637-6647, 2001); Luo etal. (Oncogene, 20: 320-328, 2001); Tomasson et al. (Blood, 93:1707-1714, 1999); Voncken et al. (, 86: 4603-4611, 1995); Andrae et al.(Mech. Dev., 107: 181-185, 2001); Reinertsen et al. (Gene Expr., 6:301-314, 1997); Huang et al. (Mol Med., 5: 129-137, 1999); Reichert etal. (Blood, 97: 1399-1403, 2001); and Huettner et al. (Nat. Genet., 24:57-60, 2000), by way of example.

Example 12 Screening Assays for Compounds that Modulate Taste ReceptorExpression or Activity

The following assays are designed to identify compounds that interactwith (e.g., bind to) a variant form of a T2R bitter taste receptor(including, but not limited to an ECD or a CD or a TMD of a variant T2Rbitter taste receptor), compounds that interact with (e.g., bind to)intracellular proteins that interact with a variant form of a T2R bittertaste receptor (including, but not limited to, a TMD or a CD of avariant form of a T2R bitter taste receptor), compounds that interferewith the interaction of a taste receptor with transmembrane orintracellular proteins involved in taste receptor-mediated signaltransduction, and to compounds which modulate the activity of a tastereceptor gene (i.e., modulate the level of gene expression) or modulatethe level of taste receptor activity of a variant form of a T2R bittertaste receptor. Assays may additionally be utilized which identifycompounds which bind to taste receptor gene regulatory sequences (e.g.,promoter sequences) and which may modulate taste receptor geneexpression. See, e.g., Platt, J Biol Chem 269:28558-28562, 1994.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to peptides, antibodies and fragmentsthereof, and other organic compounds (e.g., peptidomimetics, smallmolecules) that bind to one or more ECDs of a variant T2R bitter tastereceptor as described herein and either mimic the activity triggered bythe natural ligand (i.e., agonists) or inhibit the activity triggered bythe natural ligand (i.e., antagonists); as well as peptides, antibodiesor fragments thereof, and other organic compounds that mimic the ECD ofa variant T2R bitter taste receptor (or a portion thereof) and bind toand “neutralize” natural ligand.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, e.g. Lam et al., Nature 354:82-84, 1991;Houghten et al., Nature 354:84-86, 1991), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang et al., Cell 72:767-778, 1993), antibodies (including,but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expressionlibrary fragments, and epitope-binding fragments thereof), and smallorganic or inorganic molecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able togain entry into an appropriate cell and affect the expression of avariant T2R bitter taste receptor gene or some other gene involved in ataste receptor signal transduction pathway (e.g., by interacting withthe regulatory region or transcription factors involved in geneexpression); or such compounds that affect the activity of a variant T2Rbitter taste receptor or the activity of some other intracellular factorinvolved in the taste receptor signal transduction pathway.

Computer modeling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate expression or activity of a variant T2R bitter taste receptor.Having identified such a compound or composition, the active sites orregions are identified. Such active sites might typically be ligandbinding sites, such as the interaction domains of a bitter molecule witha variant T2R bitter taste receptor itself, or the interaction domainsof a bitter molecule with a specific allelic variant T2R bitter tastereceptor isoform in comparison to the interaction domains of thatmolecule with another isoform of the same or a different T2R bittertaste receptor (to reproduce the effect of an amino acid substitutionsuch as the alanine substitution in the PTC gene (T2R38) for designingbitter taste blockers, or to reproduce the effect of the prolinesubstitution in the PTC gene for designing bitter taste mimics).

The active site can be identified using methods known in the artincluding, for example, from the amino acid sequences of peptides, fromthe nucleotide sequences of nucleic acids, or from study of complexes ofthe relevant compound or composition with its natural ligand. In thelatter case, chemical methods can be used to find the active site byfinding where on the factor the complexed ligand is found. Next, thethree dimensional geometric structure of the active site is determined.This can be done by known methods can determine a complete molecularstructure. On the other hand, solid or liquid phase NMR can be used todetermine certain intra-molecular distances. Any other experimentalmethod of structure determination can be used to obtain partial orcomplete geometric structures, such as high resolution electronmicroscopy. The geometric structures may be measured with a complexedligand, natural or artificial, which may increase the accuracy of theactive site structure determined. In another embodiment, the structureof the specified taste receptor is compared to that of a “variant” ofthe specified taste receptor and, rather than solve the entirestructure, the structure is solved for the protein domains that arechanged.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modeling can be used to complete thestructure or improve its accuracy. Any recognized modeling method may beused, including parameterized models specific to particular biopolymerssuch as proteins or nucleic acids, molecular dynamics models based oncomputing molecular motions, statistical mechanics models based onthermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual but is preferably computer assisted. Thesecompounds found from this search are potential variant T2R bitter tastereceptor modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modeling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

In another embodiment, the structure of a specified allelic tastereceptor (the reference form) is compared to that of a variant tastereceptor (encoded by a different allele of the same specified receptor).Then, potential bitter taste inhibitors are designed that bring about astructural change in the reference form so that it resembles the variantform Or, potential bitter taste mimics are designed that bring about astructural change in the variant form so that it resembles anothervariant form, or the form of the reference receptor.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites ofbitter compounds, various variants of the T2R bitter taste receptorsdescribed herein, and related transduction and transcription factorswill be apparent to those of skill in the art.

Examples of molecular modeling systems are the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific-proteins, such as Rotivinen et al. Acta Pharmaceutical Fennica97:159-166, 1988; Ripka, New Scientist 54-57, 1988; McKinaly andRossmann, Annu Rev Pharmacol Toxicol 29:111-122, 1989; Perry and Davies,OSAR: Quantitative Structure-Activity Relationships in Drug Design pp.189-193, 1989 (Alan R. Liss, Inc.); Lewis and Dean, Proc R Soc Lond236:125-140 and 141-162, 1989; and, with respect to a model receptor fornucleic acid components, Askew et al., J Am Chem Soc 111: 1082-1090,1989. Other computer programs that screen and graphically depictchemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of a variantT2R bitter taste receptor gene product, and for designing bitter tasteblockers and mimics.

Example 13 In Vitro Screening Assays for Compounds that Bind to aVariant T2R Taste Receptor

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) a variant T2R bitter taste receptor(including, but not limited to, an ECD, or a TMD, or a CD of a variantT2R bitter taste receptor). Compounds identified may be useful, forexample, in modulating the activity of “wild type” and/or “variant” T2Rbitter taste receptor gene products; may be useful in elaborating thebiological function of taste receptors; may be utilized in screens foridentifying compounds that disrupt normal T2R bitter taste receptorinteractions; or may in themselves disrupt such interactions.

The principle of assays used to identify compounds that bind to avariant T2R bitter taste receptor involves preparing a reaction mixtureof a variant T2R bitter taste receptor polypeptide and a test compoundunder conditions and for a time sufficient to allow the two componentsto interact and bind, thus forming a complex which can be removed and/ordetected in the reaction mixture. The variant T2R bitter taste receptorspecies used can vary depending upon the goal of the screening assay.For example, where agonists or antagonists are sought, the full lengthvariant T2R bitter taste receptor, or a soluble truncated tastereceptor, e.g. in which a TMD and/or a CD is deleted from the molecule,a peptide corresponding to an ECD or a fusion protein containing avariant T2R bitter taste receptor ECD fused to a protein or polypeptidethat affords advantages in the assay system (e.g., labeling, isolationof the resulting complex, etc.) can be utilized. Where compounds thatinteract with the cytoplasmic domain are sought to be identified,peptides corresponding to a variant T2R bitter taste receptor CD andfusion proteins containing a variant T2R bitter taste receptor CD can beused.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the variantT2R bitter taste receptor protein, polypeptide, peptide or fusionprotein or the test substance onto a solid phase and detecting tastereceptor/test compound complexes anchored on the solid phase at the endof the reaction. In one embodiment of such a method, the taste receptorreactant may be anchored onto a solid surface, and the test compound,which is not anchored, may be labeled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for a variant T2Rbitter taste receptor protein, polypeptide, peptide or fusion protein orthe test compound to anchor any complexes formed in solution, and alabeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

Alternatively, cell-based assays, membrane vesicle-based assays andmembrane fraction-based assays can be used to identify compounds thatinteract with a variant T2R bitter taste receptor. To this end, celllines that express a variant T2R bitter taste receptor (or combinationthereof) or cell lines (e.g., COS cells, CHO cells, HEK293 cells, etc.)have been genetically engineered to express variant T2R bitter tastereceptor (e.g., by transfection or transduction of taste receptor DNA)can be used. Interaction of the test compound with, for example, an ECDor a CD of a variant T2R bitter taste receptor expressed by the hostcell can be determined by comparison or competition with a bittercompound or analog thereof, such as PTC.

A variant T2R bitter taste receptor polypeptide (such as those describedherein) may be employed in a screening process for compounds which bindthe receptor and which activate (agonists) or inhibit activation(antagonists) of the receptor polypeptide of the present invention.Thus, polypeptides described herein may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See Coligan et al.Current Protocols in Immunology 1 (2): Chapter 5, 1991.

In general, such screening procedures involve providing appropriatecells which express a receptor polypeptide of the present invention onthe surface thereof. Such cells include cells from mammals, insects,yeast, and bacteria. In particular, a polynucleotide encoding thereceptor of the present invention is employed to transfect cells tothereby express a variant T2R bitter taste receptor. The expressedreceptor is then contacted with a test compound to observe binding,stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores that aretransfected to express a variant T2R bitter taste receptor. Such ascreening technique is described in PCT WO 92/01810, published Feb. 6,1992, and incorporated herein by reference. Such an assay may beemployed to screen for a compound which inhibits activation of areceptor of the present invention by contacting the melanophore cellswhich encode the receptor with both a receptor ligand, such as PTC oranother bitter compound, and a compound to be screened. Inhibition ofthe signal generated by the ligand indicates that a compound is apotential antagonist for the receptor, i.e., inhibits activation of thereceptor.

The technique may also be employed for screening of compounds whichactivate a receptor of the present invention by contacting such cellswith compounds to be screened and determining whether such compoundgenerates a signal, i.e., activates the receptor.

Other screening techniques include the use of cells which express avariant T2R bitter taste receptor (for example, transfected CHO cells)in a system which measures extracellular pH changes caused by receptoractivation. In this technique, compounds may be contacted with cellsexpressing a receptor polypeptide of the present invention. A secondmessenger response, e.g., signal transduction or pH changes, is thenmeasured to determine whether the potential compound activates orinhibits the receptor.

Another screening technique involves expressing a variant T2R bittertaste receptor in which the receptor is linked to phospholipase C or D.Representative examples of such cells include, but are not limited to,endothelial cells, smooth muscle cells, and embryonic kidney cells. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

Another method involves screening for compounds which are antagonists,and thus inhibit activation of a receptor polypeptide of the presentinvention by determining inhibition of binding of labeled ligand, suchas PTC or another bitter compound, to cells which have the receptor onthe surface thereof, or cell membranes containing the receptor. Such amethod involves transfecting a eukaryotic cell with a DNA encoding avariant T2R bitter taste receptor such that the cell expresses thereceptor on its surface, or using of eukaryotic cells that express thereceptor of the present invention on their surface (or using aeukaryotic cell that expresses the receptor on its surface). The cell isthen contacted with a potential antagonist in the presence of a labeledform of a ligand, such as PTC or another bitter compound. The ligand canbe labeled, e.g., by radioactivity. The amount of labeled ligand boundto the receptors is measured, e.g., by measuring radioactivityassociated with transfected cells or membrane from these cells. If thecompound binds to the receptor, the binding of labeled ligand to thereceptor is inhibited as determined by a reduction of labeled ligandthat binds to the receptors. This method is called a binding assay.

Another such screening procedure involves the use of eukaryotic cells,which are transfected to express the receptor of the present invention,or use of eukaryotic cells that express the receptor of the presentinvention on their surface. The cells are loaded with an indicator dyethat produces a fluorescent signal when bound to calcium, and the cellsare contacted with a test substance and a receptor agonist, such as PTCor another bitter compound. Any change in fluorescent signal is measuredover a defined period of time using, for example, a fluorescencespectrophotometer or a fluorescence imaging plate reader. A change inthe fluorescence signal pattern generated by the ligand indicates that acompound is a potential antagonist (or agonist) for the receptor.

Another such screening procedure involves use of eukaryotic cells, whichare transfected to express the receptor of the present invention (or useof eukaryotic cells that express the receptor of the present invention),and which are also transfected with a reporter gene construct that iscoupled to activation of the receptor (for example, luciferase orbeta-galactosidase behind an appropriate promoter). The cells arecontacted with a test substance and a receptor agonist, such as PTC oranother bitter compound, and the signal produced by the reporter gene ismeasured after a defined period of time. The signal can be measuredusing a luminometer, spectrophotometer, fluorimeter, or other suchinstrument appropriate for the specific reporter construct used.Inhibition of the signal generated by the ligand indicates that acompound is a potential antagonist for the receptor.

Another such screening technique for antagonists or agonists involvesintroducing RNA encoding a PTC taste receptor into Xenopus oocytes totransiently express the receptor. The receptor expressing oocytes arethen contacted with a receptor ligand, such as PTC, and a compound to bescreened. Inhibition or activation of the receptor is then determined bydetection of a signal, such as, cAMP, calcium, proton, or other ions.

Another such technique of screening for antagonists or agonists involvesdetermining inhibition or stimulation of T2R taste receptor-mediatedcAMP and/or adenylate cyclase accumulation or diminution. Such a methodinvolves transiently or stably transfecting a eukaryotic cell with avariant T2R bitter taste receptor to express the receptor on the cellsurface (or using a eukaryotic cell that expresses the receptor of thepresent invention on its surface). The cell is then exposed to potentialantagonists in the presence of ligand, such as PTC or another bittercompound. The amount of cAMP accumulation is then measured, for example,by radio-immuno or protein binding assays (for example using Flashplatesor a scintillation proximity assay). Changes in cAMP levels can also bedetermined by directly measuring the activity of the enzyme, adenylylcyclase, in broken cell preparations. If the potential antagonist bindsthe receptor, and thus inhibits taste receptor binding, the levels ofvariant T2R bitter taste receptor-mediated cAMP, or adenylate cyclaseactivity, will be reduced or increased. Additional techniques forexamining the activity of G-protein receptor pathways, and componentstherein, are known to those of ordinary skill in the art.

Example 14 Assays for Intracellular Proteins that Interact with aVariant T2R Bitter Taste Receptor

Any method suitable for detecting protein-protein interactions may beemployed for identifying transmembrane proteins or intracellularproteins that interact with a variant T2R bitter taste receptor. Amongthe traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates and a variant T2R bitter taste receptor toidentify proteins in the lysate that interact with the PTC tastereceptor. For these assays, a variant T2R bitter taste receptorcomponent used can be a full length taste receptor, a soluble derivativelacking the membrane-anchoring region (e.g., a truncated taste receptorin which all TMDs are deleted resulting in a truncated moleculecontaining ECDs fused to CDs), a peptide corresponding to a CD or afusion protein containing a CD of PTC taste receptor.

Once isolated, such an intracellular protein can be identified and can,in turn, be used, in conjunction with standard techniques, to identifyproteins with which it interacts. For example, at least a portion of theamino acid sequence of an intracellular protein which interacts with thevariant T2R bitter taste receptor can be ascertained using techniqueswell known to those of skill in the art, such as via the Edmandegradation technique. See, e.g., Creighton Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N.Y., pp. 34-49, 1983. Theamino acid sequence obtained may be used as a guide for the generationof oligonucleotide mixtures that can be used to screen for genesequences encoding such intracellular proteins. Screening may beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and thescreening are well known. See, e.g. Ausubel et al. Current Protocols inMolecular Biology Green Publishing Associates and Wiley Interscience,N.Y., 1989; and Innis et al., eds. PCR Protocols: A Guide to Methods andApplications Academic Press, Inc., New York, 1990.

Additionally, methods may be employed which result in the simultaneousidentification of genes, which encode the transmembrane or intracellularproteins interacting with a variant T2R bitter taste receptor. Thesemethods include, for example, probing expression libraries, in a mannersimilar to the well known technique of antibody probing of λgt11libraries, using labeled PTC taste receptor protein, or a variant T2Rbitter taste receptor polypeptide, peptide or fusion protein, e.g., avariant T2R bitter taste receptor polypeptide or PTC taste receptordomain fused to a marker (e.g., an enzyme, fluor, luminescent protein,or dye), or an Ig-Fc domain.

One method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,PNAS USA 88:9578-9582, 1991) and is commercially available from Clontech(Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to avariant T2R bitter taste receptor nucleotide sequence encoding a variantT2R bitter taste receptor, a variant T2R bitter taste receptorpolypeptide, peptide or fusion protein, and the other plasmid consistsof nucleotides encoding the transcription activator protein's activationdomain fused to a cDNA encoding an unknown protein which has beenrecombined into this plasmid as part of a cDNA library. The DNA-bindingdomain fusion plasmid and the cDNA library are transformed into a strainof the yeast Saccharomyces cerevisiae that contains a reporter gene(e.g., HBS or lacZ) whose regulatory region contains the transcriptionactivator's binding site. Either hybrid protein alone cannot activatetranscription of the reporter gene: the DNA-binding domain hybrid cannotbecause it does not provide activation function and the activationdomain hybrid cannot because it cannot localize to the activator'sbinding sites. Interaction of the two hybrid proteins reconstitutes thefunctional activator protein and results in expression of the reportergene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, PTC tastereceptor may be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait variant T2R bittertaste receptor gene product fused to the DNA-binding domain arecotransformed into a yeast reporter strain, and the resultingtransformants are screened for those that express the reporter gene. Forexample, and not by way of limitation, a bait variant T2R bitter tastereceptor gene sequence, such as the open reading frame of variant T2Rbitter taste receptor (or a domain of a taste receptor) can be clonedinto a vector such that it is translationally fused to the DNA encodingthe DNA-binding domain of the GAL4 protein. These colonies are purifiedand the library plasmids responsible for reporter gene expression areisolated. DNA sequencing is then used to identify the proteins encodedby the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait variant T2R bitter taste receptor gene product are to be detectedcan be made using methods routinely practiced in the art. According tothe particular system described herein, for example, the cDNA fragmentscan be inserted into a vector such that they are translationally fusedto the transcriptional activation domain of GAL4. This library can beco-transformed along with the bait PTC taste receptor gene-GAL4 fusionplasmid into a yeast strain, which contains a lacZ gene driven by apromoter that contains GAL4 activation sequence. A cDNA encoded protein,fused to GAL4 transcriptional activation domain, that interacts withbait PTC taste receptor gene product will reconstitute an active GAL4protein and thereby drive expression of the HIS3 gene. Colonies, whichexpress HIS3, can be detected by their growth on Petri dishes containingsemi-solid agar based media lacking histidine. The cDNA can then bepurified from these strains, and used to produce and isolate the baitPTC taste receptor gene-interacting protein using techniques routinelypracticed in the art.

Example 15 Assays for Compounds that Interfere with TasteReceptor/Intracellular or Taste Receptor/Transmembrane MacromoleculeInteraction

The macromolecules that interact with a variant T2R bitter tastereceptor are referred to, for purposes of this discussion, as “bindingpartners”. These binding partners are likely to be involved in a variantT2R bitter taste receptor signal transduction pathway, and therefore, inthe role of taste receptors and taste receptor variants in bittertasting. Therefore, it is desirable to identify compounds that interferewith or disrupt the interaction of such binding partners with variantand/or normal T2R bitter taste receptor, which may be useful inregulating the activity of variant T2R bitter taste receptors andcontrol the sensitivity to bitter tastes associated with certain tastereceptor activity.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between a variant T2R bitter tastereceptor and its binding partner or partners involves preparing areaction mixture containing variant T2R bitter taste receptor protein,polypeptide, peptide or fusion protein as described above, and thebinding partner under conditions and for a time sufficient to allow thetwo to interact and bind, thus forming a complex. In order to test acompound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of a variant T2R bitter taste receptor moietyand its binding partner. Control reaction mixtures are incubated withoutthe test compound or with a placebo. The formation of any complexesbetween a variant T2R bitter taste receptor moiety and the bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of a variantT2R bitter taste receptor and the binding partner. Additionally, complexformation within reaction mixtures containing the test compound andreference T2R bitter taste receptor variant may also be compared tocomplex formation within reaction mixtures containing the test compoundand a different allelic or other variant of the same T2R taste receptor.This comparison may be important in those cases wherein it is desirableto identify compounds that disrupt interactions of reference but notvariant T2R taste receptors, or differentially disrupt interactionsbetween different variant T2R taste receptors.

The assay for compounds that interfere with the interaction of a variantT2R bitter taste receptor and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either a variant T2R bitter taste receptor moiety product orthe binding partner onto a solid phase and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction by competition can beidentified by conducting the reaction in the presence of the testsubstance; i.e., by adding the test substance to the reaction mixtureprior to or simultaneously with a variant T2R bitter taste receptormoiety and interactive binding partner. Alternatively, test compoundsthat disrupt preformed complexes, e.g., compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either a variant T2R bitter tastereceptor moiety or the interactive binding partner, is anchored onto asolid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtiter plates are convenientlyutilized. The anchored species may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished simplyby coating the solid surface with a solution of a variant T2R bittertaste receptor gene product or binding partner and drying.Alternatively, an immobilized antibody specific for the species to beanchored may be used to anchor the species to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of a variant T2R bittertaste receptor moiety and the interactive binding partner is prepared inwhich either a variant T2R bitter taste receptor or its binding partnersis labeled, but the signal generated by the label is quenched due toformation of the complex (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein, which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the speciesfrom the preformed complex will result in the generation of a signalabove background. In this way, test substances, which disrupt PTC tastereceptor/intracellular binding partner interaction can be identified.

In a particular embodiment, a variant T2R bitter taste receptor fusioncan be prepared for immobilization. For example, a variant T2R bittertaste receptor or a peptide fragment, e.g., corresponding to a CD, canbe fused to a glutathione-S-transferase (GST) gene using a fusionvector, such as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive bindingpartner can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above. Thisantibody can be labeled with the radioactive isotope ¹²⁵I, for example,by methods routinely practiced in the art. In a heterogeneous assay,e.g., the GST-taste receptor fusion protein can be anchored toglutathione-agarose beads. The interactive binding partner can then beadded in the presence or absence of the test compound in a manner thatallows interaction and binding to occur. At the end of the reactionperiod, unbound material can be washed away, and the labeled monoclonalantibody can be added to the system and allowed to bind to the complexedcomponents. The interaction between a variant T2R bitter taste receptorgene product and the interactive binding partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-taste receptor fusion protein and the interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of inhibition of a variant T2R bittertaste receptor/binding partner interaction can be detected by adding thelabeled antibody and measuring the radioactivity associated with thebeads.

In another embodiment, these same techniques can be employed usingpeptide fragments that correspond to the binding domains of a variantT2R bitter taste receptor and/or the interactive or binding partner (incases where the binding partner is a protein), in place of one or bothof the full length proteins. Any number of methods routinely practicedin the art can be used to identify and isolate the binding sites. Thesemethods include, but are not limited to, mutagenesis of the geneencoding one of the proteins and screening for disruption of binding ina co-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a variant T2R bitter tastereceptor gene product can be anchored to a solid material as described,above, by making a GST-taste receptor fusion protein and allowing it tobind to glutathione agarose beads. The interactive binding partner canbe labeled with a radioactive isotope, such as ³⁵S, and cleaved with aproteolytic enzyme such as trypsin. Cleavage products can then be addedto the anchored GST-taste receptor fusion protein and allowed to bind.After washing away unbound peptides, labeled bound material.,representing the intracellular binding partner binding domain, can beeluted, purified, and analyzed for amino acid sequence by well-knownmethods. Peptides so identified can be produced synthetically or fusedto appropriate facilitative proteins using recombinant DNA technology.

Example 16 Assays for Identification of Compounds that Modulate BitterTastes

Compounds, including but not limited to compounds identified via assaytechniques such as those described above, can be tested for the abilityto modulate bitter tastes. The assays described above can identifycompounds that affect variant T2R bitter taste receptor activity (e.g.,compounds that bind to a variant T2R bitter taste receptor, inhibitbinding of the natural ligand, and either activate signal transduction(agonists) or block activation (antagonists), and compounds that bind toa ligand of a variant T2R bitter taste receptor and neutralize ligandactivity); or compounds that affect variant T2R bitter taste receptorgene activity (by affecting T2R bitter taste receptor gene expression,including molecules, e.g., proteins or small organic molecules, thataffect or interfere with events so that expression of the full lengthvariant or wild-type T2R bitter taste receptor can be modulated).However, it should be noted that the assays described can also identifycompounds that modulate variant T2R bitter taste receptor signaltransduction (e.g., compounds which affect downstream signaling events,such as inhibitors or enhancers of protein kinases or phosphatasesactivities which participate in transducing the signal activated bybinding of a bitter compound (e.g., PTC) to a variant T2R bitter tastereceptor). The identification and use of such compounds which affectanother step in a variant T2R bitter taste receptor signal transductionpathway in which a variant T2R bitter taste receptor and/or variant T2Rbitter taste receptor gene product is involved and, by affecting thissame pathway may modulate the effect of variant T2R bitter tastereceptor on the sensitivity to bitter tastes are within the scope of theinvention. Such compounds can be used as part of a therapeutic methodfor modulating bitter tastes.

Cell-based systems, membrane vesicle-based systems and membranefraction-based systems can be used to identify compounds that may act tomodulate bitter tastes. Such cell systems can include, for example,recombinant or non-recombinant cells, such as cell lines, which expressthe PTC taste receptor gene. In addition, expression host cells (e.g.,COS cells, CHO cells, HEK293 cells) genetically engineered to express afunctional variant T2R bitter taste receptor and to respond toactivation by the natural ligand, e.g., as measured by a chemical orphenotypic change, induction of another host cell gene, change in ionflux (e.g., Ca²⁺), phosphorylation of host cell proteins, etc., can beused as an end point in the assay.

In utilizing such cell systems, cells may be exposed to a compoundsuspected of exhibiting an ability to modulate bitter tastes, at asufficient concentration and for a time sufficient to elicit such amodulation in the exposed cells. After exposure, the cells can beassayed to measure alterations in the expression of a variant T2R bittertaste receptor gene, e.g., by assaying cell lysates for PTC tastereceptor mRNA transcripts (e.g., by Northern analysis) or for variantT2R bitter taste receptor protein expressed in the cell; compounds whichregulate or modulate expression of a variant T2R bitter taste receptorgene are good candidates as therapeutics. Alternatively, the cells areexamined to determine whether one or more cellular phenotypes has beenaltered to resemble a taster or nontaster type. Still further, theexpression and/or activity of components of the signal transductionpathway of which a variant T2R bitter taste receptor is a part, or theactivity of a T2R bitter taste receptor signal transduction pathwayitself can be assayed.

For example, after exposure, the cell lysates can be assayed for thepresence of phosphorylation of host cell proteins, as compared tolysates derived from unexposed control cells. The ability of a testcompound to inhibit phosphorylation of host cell proteins in these assaysystems indicates that the test compound alters signal transductioninitiated by taste receptor activation. The cell lysates can be readilyassayed using a Western blot format; i.e., the host cell proteins areresolved by gel electrophoresis, transferred and probed using adetection antibody (e.g., an antibody labeled with a signal generatingcompound, such as radiolabel, fluor, enzyme, etc.), see, e.g. Glenney etal., J Immunol Methods 109:277-285, 1988; Frackelton et al., Mol CellBiol 3:1343-1352, 1983. Alternatively, an ELISA format could be used inwhich a particular host cell protein involved in the taste receptorsignal transduction pathway is immobilized using an anchoring antibodyspecific for the target host cell protein, and the presence or absenceof a phosphorylated residue on the immobilized host cell protein isdetected using a labeled antibody. (See, e.g. King et al., Life Sci53:1465-1472, 1993).

In yet another approach, ion flux, such as calcium ion flux, can bemeasured as an end point for PTC taste receptor stimulated signaltransduction. Calcium flux can be measured, for instance, usingcalcium-sensitive dyes or probes, many of which are known to those ofordinary skill in the art. Examples of ion sensitive fluorophoresinclude, but are not limited to, bis-(1,3-dibutylbarbituricacid)trimethine oxonol (DiBAC4(3) (B-438), Quin-2 (AM Q-1288), Fura-2(AM F-1225), Indo-1 (AM I-1226), Fura-3 (AM F-1228), Fluo-3 (AM F-1241),Rhod-2, (AM R-1244), BAPTA (AM B-1205), 5,5′-dimethyl BAPTA (AM D-1207),4,4′-difluoro BAPTA (AM D-1216), 5,5′-difluoro BAPTA (AM D-1209),5,5′-dibromo BAPTA (AM D-1213), Calcium Green (C-3011), Calcium Orange(C-3014), Calcium Crimson (C-3017), Fura-5 (F-3023), Fura-Red (F-3020),SBFI (S-1262), PBFI (P-1265), Mag-Fura-2 (AM M-1291), Mag-Indo-1 (AMM-1294), Mag-Quin-2 (AM M-1299), Mag-Quin-1 (AM M-1297), SPQ (M-440),SPA (S-460), Calcien (Fluorescein-bis(methyliminodiacetic acid);Fluorexon), and Quin-2(2-{[2-Bis-(carboxymethyl)amino-5-methylphenoxy]-methyl}-6-methoxy-8-bis-(carboxymethyl)aminoquinolinetetrapotassium salt).

Example 17 Other Assays for Modulators of Variant T2R Bitter TasteReceptors

A. Assays for Taste Receptor Protein Activity

T2R bitter taste receptor family members are G-protein coupled receptorsthat participate in taste transduction, e.g., bitter taste transduction.The activity of a T2R bitter taste receptor protein variants can beassessed using a variety of in vitro and in vivo assays to determinefunctional, chemical, and physical effects, e.g., measuring ligandbinding (e.g., radioactive ligand binding), second messengers (e.g.,cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels,transcription levels, neurotransmitter levels, and the like.Furthermore, such assays can be used to test for inhibitors andactivators of identified T2R bitter taste receptor family membervariants. Modulators can also be genetically altered versions of tastereceptors. Such modulators of taste transduction activity are useful forcustomizing taste, for example to modify the detection of bitter tastes.

Modulators of a T2R bitter taste receptor protein variant activity aretested using taste receptor polypeptides as described herein, eitherrecombinant or naturally occurring. The protein can be isolated,expressed in a cell, expressed in a membrane derived from a cell,expressed in tissue or in an animal, either recombinant or naturallyoccurring. For example, tongue slices, dissociated cells from a tongue,transformed cells, or membranes can be used. Modulation is tested usingone of the in vitro or in vivo assays described herein. Tastetransduction can also be examined in vitro with soluble or solid statereactions, using a full-length taste receptor or a chimeric moleculesuch as an extracellular domain or transmembrane domain, or combinationthereof, of a taste receptor variant covalently linked to a heterologoussignal transduction domain, or a heterologous extracellular domainand/or transmembrane domain covalently linked to the transmembraneand/or cytoplasmic domain of a T2R bitter taste receptor proteinvariant. Furthermore, ligand-binding domains of the protein of interestcan be used in vitro in soluble or solid state reactions to assay forligand binding. In numerous embodiments, a chimeric receptor will bemade that comprises all or part of a T2R bitter taste receptor proteinvariant as well an additional sequence that facilitates the localizationof the taste receptor to the membrane, such as a rhodopsin, e.g., anN-terminal fragment of a rhodopsin protein.

Ligand binding a T2R bitter taste receptor protein variant, a domain, orchimeric protein can be tested in solution, in a bilayer membrane,attached to a solid phase, in a lipid monolayer, or in vesicles. Bindingof a modulator can be tested using, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index)hydrodynamic (e.g., shape), chromatographic, or solubility properties.

Receptor-G-protein interactions can also be examined. For example,binding of the G-protein to the receptor or its release from thereceptor can be examined. For example, in the absence of GTP, anactivator will lead to the formation of a tight complex of a G protein(all three known subunits) with the receptor. This complex can bedetected in a variety of ways, as noted above. Such an assay can bemodified to search for inhibitors, e.g., by adding an activator to thereceptor and G protein in the absence of GTP, which form a tightcomplex, and then screen for inhibitors by looking at dissociation ofthe receptor-G protein complex. In the presence of GTP, release of theknown alpha subunit of the G protein from the other two known G proteinsubunits serves as a criterion of activation.

In a convenient embodiment, T2R bitter taste receptor proteinvariant-gustducin interactions are monitored as a function of tastereceptor activation. One taste-cell specific G protein that has beenidentified is called gustducin (McLaughlin et al. Nature 357:563-569,1992). Such ligand dependent coupling of taste receptors with gustducincan be used as a marker to identify modifiers of the T2R bitter tastereceptor protein variant

An activated or inhibited G-protein will in turn alter the properties oftarget enzymes, channels, and other effector proteins. The classicexamples are the activation of cGMP phosphodiesterase by transducin inthe visual system, adenylate cyclase by the stimulatory G-protein,phospholipase C by Gq and other cognate G proteins, and modulation ofdiverse channels by Gi and other G proteins. Downstream consequences canalso be examined such as generation of diacyl glycerol and IP3 byphospholipase C, and in turn, for calcium mobilization by IP3.

In a convenient embodiment, a T2R bitter taste receptor protein variantis expressed in a eukaryotic cell as a chimeric receptor with aheterologous, chaperone sequence that facilitates its maturation andtargeting through the secretory pathway. In a preferred embodiment, theheterologous sequence is a rhodopsin sequence, such as an N-terminalleader of a rhodopsin. Such chimeric taste receptors can be expressed inany eukaryotic cell, such as HEK293 cells. Preferably, the cellscomprise a functional G protein, e.g., Gα15, that is capable of couplingthe chimeric receptor to an intracellular signaling pathway or to asignaling protein such as phospholipase Cβ. Activation of such chimericreceptors in such cells can be detected using any standard method, suchas by detecting changes in intracellular calcium by detecting FURA-2dependent fluorescence in the cell.

An activated G-protein coupled receptor (GPCR) becomes a substrate forkinases that phosphorylate the C-terminal tail of the receptor (andpossibly other sites as well). Thus, activators will promote thetransfer of ³²P from gamma-labeled GTP to the receptor, which can beassayed with a scintillation counter. The phosphorylation of theC-terminal tail will promote the binding of arrestin-like proteins andwill interfere with the binding of G-proteins. The kinase/arrestinpathway plays a key role in the desensitization of many GPCR receptors.For example, compounds that modulate the duration a taste receptor staysactive would be useful as a means of prolonging a desired taste orcutting off an unpleasant one. For a general review of GPCR signaltransduction and methods of assaying signal transduction, see, e.g.Methods in Enzymology, vols. 237 and 238 (1994) and volume 96 (1983);Bourne et al., Nature 10:349:117-127, 1991; Bourne et al., Nature348:125-132, 1990; Pitcher et al., Annu Rev Biochem 67:653-692, 1998.

Samples or assays that are treated with a potential T2R bitter tastereceptor protein variant inhibitor or activator are compared to controlsamples without the test compound, to examine the extent of modulation.Such assays may be carried out in the presence of a bitter tastant thatis known to activate the particular receptor, and modulation of thebitter-tastant-dependent activation monitored. Control samples(untreated with activators or inhibitors) are assigned a relative T2Rbitter taste receptor protein activity value of 100. Inhibition of a T2Rbitter taste receptor protein variant is achieved when the T2R bittertaste receptor protein variant activity value relative to the control isabout 90%, optionally 50%, optionally 25-0%. Activation of a T2R bittertaste receptor protein variant is achieved when the T2R bitter tastereceptor protein variant activity value relative to the control is 110%,optionally 150%, 200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a T2R bitter taste receptor protein variant. One means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g. Ackerman et al.,New Engl J Med 336:1575-1595, 1997). Whole cell currents areconveniently determined using the standard methodology (see, e.g., Hamilet al., Pflugers Archiv 391:85, 1981). Other known assays include:radiolabeled ion flux assays and fluorescence assays usingvoltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J MembraneBiol 88:67-75, 1988; Gonzales & Tsien, Chem Biol 4:269-277, 1997; Danielet al., J Pharmacol Meth 25:185-193, 1991; Holevinsky et al., J MembraneBiology 137:59-70, 1994). Generally, the compounds to be tested arepresent in the range from 1 pM to 100 mM.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3, cGMP, orcAMP.

Convenient assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors asnegative or positive controls to assess activity of tested compounds. Inassays for identifying modulatory compounds (e.g., agonists,antagonists), changes in the level of ions in the cytoplasm or membranevoltage will be monitored using an ion sensitive or membrane voltagefluorescent indicator, respectively. Among the ion-sensitive indicatorsand voltage probes that may be employed are those disclosed in theMolecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al. PNAS USA 88:10049-10053, 1991). Such promiscuousG-proteins allow coupling of a wide range of receptors.

Receptor activation typically initiates subsequent intracellular events,e.g. increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol triphosphate(IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine Nature, 312:315-321, 1984). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g. Altenhofen et al., PNAS USA 88:9868-9872, 1991;and Dhallan et al., Nature 347:184-187, 1990). In cases where activationof the receptor results in a decrease in cyclic nucleotide levels, itmay be preferable to expose the cells to agents that increaseintracellular cyclic nucleotide levels, e.g., forskolin, prior to addinga receptor-activating compound to the cells in the assay. Cells for thistype of assay can be made by co-transfection of a host cell with DNAencoding a cyclic nucleotide-crated ion channel, GPCR phosphatase andDNA encoding a receptor (e.g., certain glutamate receptors, muscarinicacetylcholine receptors, dopamine receptors, serotonin receptors, andthe like), which, when activated, causes a change in cyclic nucleotidelevels in the cytoplasm.

In a convenient embodiment, a T2R bitter taste receptor protein variantactivity is measured by expressing a T2R bitter taste receptor proteinvariant gene in a heterologous cell with a promiscuous G-protein thatlinks the receptor to a phospholipase C signal transduction pathway(see, Offermanns & Simon, J Biol Chem 270:15175-15180, 1995). Optionallythe cell line is HEK293 (which does not naturally express PTC tastereceptor genes and the promiscuous G-protein is Gα15 (Offermanns &Simon, 1995). Modulation of taste transduction is assayed by measuringchanges in intracellular Ca²⁺ levels, which change in response tomodulation of the a T2R bitter taste receptor protein variant signaltransduction pathway via administration of a molecule that associateswith a T2R bitter taste receptor protein variant. Changes in Ca²⁺ levelsare optionally measured using fluorescent Ca²⁺ indicator dyes andfluorometric imaging. Examples of ion sensitive dyes and probes include,but are not limited to, bis-(1,3-dibutylbarbituric acid)trimethineoxonol (DiBAC4(3) (B-438), Quin-2 (AM Q-1288), Fura-2 (AM F-1225),Indo-1 (AM I-1226), Fura-3 (AM F-1228), Fluo-3 (AM F-1241), Rhod-2, (AMR-1244), BAPTA (AM B-1205), 5,5′-dimethyl BAPTA (AM D-1207),4,4′-difluoro BAPTA (AM D-1216), 5,5′-difluoro BAPTA (AM D-1209),5,5′-dibromo BAPTA (AM D-1213), Calcium Green (C-3011), Calcium Orange(C-3014), Calcium Crimson (C-3017), Fura-5 (F-3023), Fura-Red (F-3020),SBFI (S-1262), PBFI (P-1265), Mag-Fura-2 (AM M-1291), Mag-Indo-1 (AMM-1294), Mag-Quin-2 (AM M-1299), Mag-Quin-1 (AM M-1297), SPQ (M440), SPA(S-460), Calcien (Fluorescein-bis(methylminodiacetic acid); Fluorexon),and Quin-2(2-{[2-Bis-(carboxymethyl)amino-5-methylphenoxy]-methyl}-6-methoxy-8-bis-(carboxymethyl)aminoquinolinetetrapotassium salt).

In one embodiment, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon(J Biol Chem 270:15175-15180, 1995), for instance, may be used todetermine the level of cAMP. Also, the method described in Felley-Boscoet al. (Am J Resp Cell and Mol Biol 11:159-164, 1994) may be used todetermine the level of cGMP. Further, an assay kit for measuring cAMPand/or cGMP is described in U.S. Pat. No. 4,115,538.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128. Briefly, the assayinvolves labeling of cells with ³H-myoinositol for 48 or more hours. Thelabeled cells are treated with a test compound for one hour. The treatedcells are lysed and extracted in chloroform-methanol-water after whichthe inositol phosphates are separated by ion exchange chromatography andquantified by scintillation counting. Fold stimulation is determined bycalculating the ratio of cpm in the presence of agonist to cpm in thepresence of buffer control. Likewise, fold inhibition is determined bycalculating the ratio of cpm in the presence of antagonist to cpm in thepresence of buffer control (which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining a T2R bitter taste receptor protein variant of interest iscontacted with a test compound for a sufficient time to effect anyinteractions, and then the level of gene expression is measured. Theamount of time to effect such interactions may be empiricallydetermined, such as by running a time course and measuring the level oftranscription as a function of time. The amount of transcription may bemeasured by using any method known to those of skill in the art to besuitable. For example, mRNA expression of the protein of interest may bedetected using northern blots or their polypeptide products may beidentified using immunoassays. Alternatively, transcription based assaysusing reporter genes may be used as described in U.S. Pat. No.5,436,128. The reporter genes can be, e.g., chloramphenicolacetyltransferase, luciferase, β-galactosidase and alkaline phosphatase.Furthermore, the protein of interest can be used as an indirect reportervia attachment to a second reporter such as green fluorescent protein(see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964, 1997).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some manneraltered the activity of the protein of interest.

B. Modulators

The compounds tested as modulators of a T2R bitter taste receptor familymember variant can be any small chemical compound, or a biologicalentity, such as a protein, sugar, nucleic acid or lipid. Alternatively,modulators can be genetically altered versions of a T2R bitter tastereceptor protein gene. Typically, test compounds will be small chemicalmolecules and peptides. Essentially any chemical compound can be used asa potential modulator or ligand in the assays of the invention, althoughmost often compounds dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St Louis,Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like.

In one convenient embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particularly chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175; Furka, Int J Pept Prot Res 37:487-493, 1991;and Houghton et al., Nature 354:84-88, 1991). Other chemistries forgenerating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptides (e.g. PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g. PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., PNAS USA90:6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J AmerChem Soc 114:6568, 1992), nonpeptidal peptidomimetics with glucosescaffolding (Hirschmann et al., J Amer Chem Soc 114:9217-9218, 1992),analogous organic syntheses of small compound libraries (Chen et al., JAmer Chem Soc 116:2661, 1994), oligocarbamates (Cho et al. 1993 Science261:1303), and/or peptidyl phosphonates (Campbell et al., J Org Chem59:658, 1994), nucleic acid libraries (see Sambrook et al. MolecularCloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y., 1989; andAusubel et al. Current Protocols in Molecular Biology Green PublishingAssociates and Wiley Interscience, N.Y., 1989), peptide nucleic acidlibraries (see, e.g. U.S. Pat. No. 5,539,083), antibody libraries (see,e.g., Vaughn et al. Nature Biotechnology 14:309-314, 1996; andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science 274:1520-1522, 1996; and U.S. Pat. No. 5,593,853), small organicmolecule libraries (see, e.g., benzodiazepines, Baum 1993 C&EN, January18, page 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones andmethathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g. 357 MPS, 390 MPS, Advanced Chem Tech, Louisville,Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, FosterCity, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment is provided soluble assays using molecules such as adomain such as a ligand binding domain, an extracellular domain, atransmembrane domain, a transmembrane domain and a cytoplasmic domain,an active site, a subunit association region, etc.; a domain that iscovalently linked to a heterologous protein to create a chimericmolecule; a T2R bitter taste receptor protein variant/isoform; or a cellor tissue expressing a T2R bitter taste receptor proteinvariant/isoform, either naturally occurring or recombinant. Anotherembodiment provides solid phase based in vitro assays in a highthroughput format, where the domain, chimeric molecule, T2R bitter tastereceptor protein variant/isoform, or cell or tissue expressing aspecific T2R bitter taste receptor variant is attached to a solid phasesubstrate. It is particularly contemplated in some embodiments thatmultiple molecules are provided in such assays, for instance, acollection of two or more T2R isoforms proteins, or fragments thereof,such as those isoforms shown in SEQ ID NOs: 4, 8, 10, 12, 14, 16, 20,22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 46, 50, 56, 58, 60, 64, 66, 68,70, 72, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 100, 102, 104, 106, 108,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 136, 140, 142, 148,150, 152, 156, 158, 162, 164, 170, 174, 176, 178, 180, 182, 184, 188,190, 192, 198, 200, 202, 204, 206, 210, 212, 214, 218, 220, 226, 228,230, 232, 234, 236, 238, 242, 244, 246, 248, 250, 252, 254, 258, 260,264.

In the high throughput assays, it is possible to screen up to severalthousand different modulators or ligands in a single day. In particular,each well of a microtiter plate can be used to run a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 100 (e.g., 96) modulators. If 1536 well plates are used, then asingle plate can easily assay from about 100-about 1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds arepossible using the integrated systems of the invention. More recently,microfluidic approaches to reagent manipulation have been developed.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non-covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.).Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis, Mo.

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, viral receptor ligands, cytokinereceptors, chemokine receptors, interleukin receptors, immunoglobulinreceptors and antibodies, the cadherein family, the integrin family, theselectin family, and the like; (see, e.g., Pigott & Power 1993 TheAdhesion Molecule Facts Book I). Similarly, toxins and venoms, viralepitopes, hormones (e.g., opiates, steroids, etc.), intracellularreceptors (e.g., which mediate the effects of various small ligands,including steroids, thyroid hormone, retinoids and vitamin D; peptides),drugs, lectins, sugars, nucleic acids (both linear and cyclic polymerconfigurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalized a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J AmChem Soc 85:2149-2154, 1963 (describing solid phase synthesis of, e.g.,peptides); Geysen et al., J Immmun Meth 102:259-274, 1987 (describingsynthesis of solid phase components on pins); Frank & Doring,Tetrahedron 44:6031-6040, 1988 (describing synthesis of various peptidesequences on cellulose disks); Fodor et al., Science 251:767-777, 1991;Sheldon et al., Clinical Chemistry 39:718-719, 1993; and Kozal et al.,Nature Medicine 2:753-759, 1996 (all describing arrays of biopolymersfixed to solid substrates). Non-chemical approaches for fixing tagbinders to substrates include other common methods, such as heat,cross-linking by UV radiation, and the like.

D. Computer-Based Assays

Yet another assay for compounds that modulate taste receptor proteinactivity involves computer assisted drug design, in which a computersystem is used to generate a three-dimensional structure of a targettaste receptor protein based on the structural information encoded byits amino acid sequence. The input amino acid sequence interactsdirectly and actively with a preestablished algorithm in a computerprogram to yield secondary, tertiary, and quaternary structural modelsof the protein. The models of the protein structure are then examined toidentify regions of the structure that have the ability to bind, e.g.,ligands. These regions are then used to identify ligands that bind tothe protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a T2R bitter tastereceptor polypeptide allelic variant into the computer system. Thenucleotide sequence encoding the polypeptide, or the amino acid sequencethereof, can be any of the allelic variant taste receptors described.The amino acid sequence represents the primary sequence or subsequenceof the protein, which encodes the structural information of the protein.At least 10 residues of the amino acid sequence (or a nucleotidesequence encoding 10 amino acids) are entered into the computer systemfrom computer keyboards, computer readable substrates that include, butare not limited to, electronic storage media (e.g., magnetic diskettes,tapes, cartridges, and chips), optical media (e.g., CD ROM), informationdistributed by internet sites, and by RAM. The three-dimensionalstructural model of the protein is then generated by the interaction ofthe amino acid sequence and the computer system, using software known tothose of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Walls potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel. An example for G-protein cell receptors is presented in Vaidehiet al. (PNAS 99:15308-15312, 2002), which is incorporated herein byreference in its entirety.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variable along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been granted, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the target taste receptor protein variant to identify ligandsthat bind to the protein. Binding affinity between the protein andligands is determined using energy terms to determine which ligands havean enhanced probability of binding to the protein.

Example 18 Pharmaceutical Preparations and Methods of Administration

Taste modulators can be administered directly to the mammalian subjectfor modulation of taste, e.g., modulation of bitter taste, in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treated,optionally the tongue or mouth. The taste modulators are administered inany suitable manner, optionally with pharmaceutically acceptablecarriers. Suitable methods of administering such modulators areavailable and well known to those of skill in the art and, although morethan one route can be used to administer a particular composition, aparticular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985).

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part of a prepared food ordrug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular taste modulators employed and the condition of the subject,as well as the body weight or surface area of the area to be treated.The size of the dose also will be determined by the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound in a particular subject.

In determining the effective amounts of the modulator to beadministered, a physician may evaluate circulating plasma levels of themodulator, modulator toxicities, and the production of anti-modulatorantibodies. In general, the dose equivalent of a modulator is from about1 ng/kg to 10 mg/kg for a typical subject.

For administration, taste modulators of the present invention can beadministered at a rate determined by the LD₅₀ of the modulator, and theside effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

Example 19 Kits

Kits are provided which contain reagents useful for determining thepresence or absence of polymorphism(s) in at least one T2R-encodingsequence, such as probes or primers specific for a T2R SNP shown in FIG.1, or a T2R haplotype allele shown in Table 7. Such kits can be usedwith the methods described herein to determine a subject's T2R genotypeor haplotype, for one or more T2R genes.

The provided kits may also include written instructions. Theinstructions can provide calibration curves or charts to compare withthe determined (e.g., experimentally measured) values.

The oligonucleotide probes and primers disclosed herein can be suppliedin the form of a kit for use in detection of a specific T2R sequence,such as a SNP or haplotype described herein, in a subject. In such akit, an appropriate amount of one or more of the oligonucleotide primersis provided in one or more containers. The oligonucleotide primers maybe provided suspended in an aqueous solution or as a freeze-dried orlyophilized powder, for instance. The container(s) in which theoligonucleotide(s) are supplied can be any conventional container thatis capable of holding the supplied form, for instance, microfuge tubes,ampoules, or bottles. In some applications, pairs of primers may beprovided in pre-measured single use amounts in individual, typicallydisposable, tubes or equivalent containers. With such an arrangement,the sample to be tested for the presence of a T2R polymorphism can beadded to the individual tubes and amplification carried out directly.

The amount of each oligonucleotide primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each oligonucleotide primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. Those of ordinary skill in the art know the amount ofoligonucleotide primer that is appropriate for use in a singleamplification reaction. General guidelines may for instance be found inInnis et al. (PCR Protocols, A Guide to Methods and Applications,Academic Press, Inc., San Diego, Calif., 1990), Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York,1989), and Ausubel et al. (In Current Protocols in Molecular Biology,Greene Publ. Assoc. and Wiley-Intersciences, 1992).

A kit may include more than two primers, in order to facilitate the invitro amplification of T2R-encoding sequences, for instance a specifictarget T2R gene or the 5′ or 3′ flanking region thereof.

In some embodiments, kits may also include the reagents necessary tocarry out nucleotide amplification reactions, including, for instance,DNA sample preparation reagents, appropriate buffers (e.g., polymerasebuffer), salts (e.g., magnesium chloride), and deoxyribonucleotides(dNTPs).

Kits may in addition include either labeled or unlabeled oligonucleotideprobes for use in detection of T2R polymorphism(s) or haplotypes. Incertain embodiments, these probes will be specific for a potentialpolymorphic site that may be present in the target amplified sequences.The appropriate sequences for such a probe will be any sequence thatincludes one or more of the identified polymorphic sites, particularlythose nucleotide positions indicated in FIG. 1, such that the sequencethe probe is complementary to a polymorphic site and the surrounding T2Rsequence. By way of example, such probes are of at least 6 nucleotidesin length, and the polymorphic site occurs at any position within thelength of the probe. It is often beneficial to use longer probes, inorder to ensure specificity. Thus, in some embodiments, the probe is atleast 8, at least 10, at least 12, at least 15, at least 20, at least 30nucleotides or longer.

It may also be advantageous to provide in the kit one or more controlsequences for use in the amplification reactions. The design ofappropriate positive control sequences is well known to one of ordinaryskill in the appropriate art. By way of example, control sequences maycomprise human (or non-human) T2R nucleic acid molecule(s) with knownsequence at one or more target SNP positions, such as those described inFIG. 1.

In some embodiments, kits may also include some or all of the reagentsnecessary to carry out RT-PCR in vitro amplification reactions,including, for instance, RNA sample preparation reagents (includinge.g., an RNase inhibitor), appropriate buffers (e.g., polymerasebuffer), salts (e.g., magnesium chloride), and deoxyribonucleotides(dNTPs).

Such kits may in addition include either labeled or unlabeledoligonucleotide probes for use in detection of the in vitro amplifiedtarget sequences. The appropriate sequences for such a probe will be anysequence that falls between the annealing sites of the two providedoligonucleotide primers, such that the sequence the probe iscomplementary to is amplified during the PCR reaction. In certainembodiments, these probes will be specific for a potential polymorphismthat may be present in the target amplified sequences.

It may also be advantageous to provide in the kit one or more controlsequences for use in the RT-PCR reactions. The design of appropriatepositive control sequences is well known to one of ordinary skill in theappropriate art.

Kits for the detection or analysis of T2R protein expression (such asover- or under-expression, or expression of a specific isoform) are alsoencompassed. Such kits may include at least one target protein specificbinding agent (e.g., a polyclonal or monoclonal antibody or antibodyfragment that specifically recognizes a T2R protein, or beneficially aspecific T2R protein isoform) and may include at least one control (suchas a determined amount of target T2R protein, or a sample containing adetermined amount of T2R protein). The T2R-protein specific bindingagent and control may be contained in separate containers.

T2R protein or isoform expression detection kits may also include ameans for detecting T2R:binding agent complexes, for instance the agentmay be detectably labeled. If the detectable agent is not labeled, itmay be detected by second antibodies or protein A for example which mayalso be provided in some kits in one or more separate containers. Suchtechniques are well known.

Additional components in specific kits may include instructions forcarrying out the assay. Instructions will allow the tester to determineT2R expression level. Reaction vessels and auxiliary reagents such aschromogens, buffers, enzymes, etc. may also be included in the kits.

Also provided are kits that allow differentiation between individualswho are homozygous versus heterozygous for specific SNPs or haplotypesof the described T2R bitter taste receptors. Examples of such kitsprovide the materials necessary to perform oligonucleotide ligationassays (OLA), as described at Nickerson et al. (Proc. Natl. Acad. Sci.USA 87:8923-8927, 1990). In specific embodiments, these kits contain oneor more microtiter plate assays, designed to detect polymorphism(s) in aT2R sequence of a subject, as described herein.

Additional components in some of these kits may include instructions forcarrying out the assay. Instructions will allow the tester to determinewhether a specified T2R allele is present, and whether it is homozygousor heterozygous. Reaction vessels and auxiliary reagents such aschromogens, buffers, enzymes, etc. may also be included in the kits.

It may also be advantageous to provide in the kit one or more controlsequences for use in the OLA reactions. The design of appropriatepositive control sequences is well known to one of ordinary skill in theappropriate art.

This disclosure provides a comprehensive collection of SNPs in bittertaste receptor genes, including sub-sets that represent conserved,non-conserved, silent, and truncation mutations in the correspondingproteins, and individual allelic sequences and haplotypes for bittertaste receptor genes. The disclosure further provides methods for usingthe corresponding allelic variants of the taste receptor genes, alone orin various combinations, to test and characterize a subject's bittertasting profile and to identify and analyze compounds that interact withand/or influence bitter tastes in different subjects and populations. Itwill be apparent that the precise details of the methods described maybe varied or modified without departing from the spirit of the describedinvention. We claim all such modifications and variations that fallwithin the scope and spirit of this disclosure, and all equivalents ofsuch.

1. An isolated T2R44 variant-specific nucleic acid molecule comprisingat least about 10 contiguous nucleotides, spanning at least one singlenucleotide polymorphism (SNP) selected from T at position 103, C atposition 423, A at position 599, G at position 649, T at position 680, Aat position 718, G at position 744, G at position 827 and A at position843 of SEQ ID NO:
 33. 2. An array, comprising the nucleic acid moleculeof claim
 1. 3. The array of claim 2, further comprising at least onenucleic acid molecule comprising at least about 10 contiguousnucleotides selected from T2R1, T2R3, T2R4, T2R5, T2R7, T2R8, T2R9,T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40, T2R41, T2R43, T2R44,T2R45, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60, and spanning atleast one SNP selected from the group consisting of: C/T at position 128(SEQ ID NO: 1); A/G at position 332 (SEQ ID NO: 1); G/A at position 422(SEQ ID NO: 1); C/T at position 616 (SEQ ID NO: 1); C/T at position 675(SEQ ID NO: 1); T/C at position 850 (SEQ ID NO: 1): C/T at position 349(SEQ ID NO: 3); C/T at position 807 (SEQ ID NO: 3); C/T at position 852(SEQ ID NO: 3); G/A at position 8 (SEQ ID NO: 5); G/C at position 9 (SEQID NO: 5); A/C at position 17 (SEQ ID NO: 5); T/C at position 20 (SEQ IDNO: 5); T/A at position 186 (SEQ ID NO: 5); C/T at position 221 (SEQ IDNO: 5); G/C at position 286 (SEQ ID NO: 5); G/A at position 512 (SEQ IDNO: 5); G/A at position 571 (SEQ ID NO: 5); G/A at position 58 (SEQ IDNO: 7); G/T at position 77 (SEQ ID NO: 7); C/T at position 235 (SEQ IDNO: 7); C/T at position 338 (SEQ ID NO: 7); G/A at position 363 (SEQ IDNO: 7); G/A at position 500 (SEQ ID NO: 7); C/A at position 638 (SEQ IDNO: 7); G/T at position 881 (SEQ ID NO: 7); A/T at position 787 (SEQ IDNO: 9); C/T at position 788 (SEQ ID NO: 9); C/A at position 828 (SEQ IDNO: 9); G/A at position 912 (SEQ ID NO: 9); A/G at position 496 (SEQ IDNO: 11); G/A at position 549 (SEQ ID NO: 11); T/C at position 829 (SEQID NO: 11); G/A at position 922 (SEQ ID NO: 11); C/A at position 201(SEQ ID NO: 13); T/A at position 450 (SEQ ID NO: 13); T/C at position560 (SEQ ID NO: 13); C/T at position 867 (SEQ ID NO: 13); C/A atposition 880 (SEQ ID NO: 13); C/T at position 910 (SEQ ID NO: 13); C/Tat position 926 (SEQ ID NO: 13); A/G at position 120 (SEQ ID NO: 15);C/T at position 467 (SEQ ID NO: 15); A/C at position 521 (SEQ ID NO:15); A/G at position 564 (SEQ ID NO: 15); G/A at position 627 (SEQ IDNO: 15); A/G at position (SEQ ID NO: 17); A/G at position 256 (SEQ IDNO: 19); G/A at position 375 (SEQ ID NO: 19); C/T at position 300 (SEQID NO: 21); G/A at position 301 (SEQ ID NO: 21); G/C at position 303(SEQ ID NO: 21); T/C at position 460 (SEQ ID NO: 21); T/G at position516 (SEQ ID NO: 21); G/A at position 665 (SEQ ID NO: 21); G/A atposition 846 (SEQ ID NO: 21); C/G at position 145 (SEQ ID NO: 23); G/Aat position 239 (SEQ ID NO: 23); C/T at position 785 (SEQ ID NO: 23);C/T at position 820 (SEQ ID NO: 23); G/A at position 886 (SEQ ID NO:23); C/T at position 578 (SEQ ID NO: 25); G/A at position 589 (SEQ IDNO: 25); G/A at position 874 (SEQ ID NO: 25); C/A at position 560 (SEQID NO: 27) G/A at position 817 of T2R40 (SEQ ID NO: 27); G/A at position189 (SEQ ID NO: 29); C/T at position 380 (SEQ ID NO: 29); T/A atposition 584 (SEQ ID NO: 29); G/C at position 104 (SEQ ID NO: 31); G/Aat position 270 (SEQ ID NO: 31); G/C at position 460 (SEQ ID NO: 31);T/G at position 510 (SEQ ID NO: 31); G/T at position 599 (SEQ ID NO:31); G/A at position 635 (SEQ ID NO: 31); C/G at position 663 (SEQ IDNO: 31); T/G at position 882 (SEQ ID NO: 31); T/C at position 883 (SEQID NO: 31); A/G at position 889 (SEQ ID NO: 31); C/T at position 103(SEQ ID NO: 33); T/C at position 423 (SEQ ID NO: 33); T/A at position484 (SEQ ID NO: 33); G/A at position 599 (SEQ ID NO: 33); C/G atposition 649 (SEQ ID NO: 33); C/T at position 680 (SEQ ID NO: 33); G/Aat position 718 (SEQ ID NO: 33); A/G at position 744 (SEQ ID NO: 33);C/G at position 827 (SEQ ID NO: 33); G/A at position 843 (SEQ ID NO:33); T/C at position 106 (SEQ ID NO: 35); T/A at position 682 (SEQ IDNO: 35); G/A at position 749 (SEQ ID NO: 35); C/T at position 862 (SEQID NO: 35); G/A at position 934 (SEQ ID NO: 37); G/A at position 920(SEQ ID NO: 37); T/G at position 842 (SEQ ID NO: 37); T/G at position756 (SEQ ID NO: 37); C/T at position 84 (SEQ ID NO 39); G/A at position94 (SEQ ID NO: 39); A/C at position 326 (SEQ ID NO: 39); T/C at position418 (SEQ ID NO: 39); A/T at position 456 (SEQ ID NO: 39); A/G atposition 673 (SEQ ID NO: 39); T/C at position 719 (SEQ ID NO: 39); G/Cat position 799 (SEQ ID NO: 39); G/A at position 885 (SEQ ID NO: 39);C/T at position 895 (SEQ ID NO: 39); A/G at position 156 (SEQ ID NO:41); C/T at position 261 (SEQ ID NO: 41); G/A at position 421 (SEQ IDNO: 41); C/A at position 429 (SEQ ID NO: 41); C/A at position 442 (SEQID NO: 41); G/A at position 516 (SEQ ID NO: 41); A/G at position 706(SEQ ID NO: 41); T/C at position 755 (SEQ ID NO: 41); G/T at position764 (SEQ ID NO: 41); A/C at position 808 (SEQ ID NO: 41); G/A atposition 608 (SEQ ID NO: 43); A/T at position 595 (SEQ ID NO: 45); andC/T at position 930 of T2R60 (SEQ ID NO: 45).
 4. The array of claim 2,further comprising at least one oligonucleotide from each T2Rhaplotype/allele selected from T2R1 (SEQ ID NO: 47), T2R3 (SEQ ID NO:53), T2R4 (SEQ ID NO: 61), T2R5 (SEQ ID NO: 73), T2R7 (SEQ ID NO: 87),T2R8 (SEQ ID NO: 97), T2R9 (SEQ ID NO: 109), T2R10 (SEQ ID NO: 131),T2R13 (SEQ ID NO: 133), T2R14 (SEQ ID NO: 137), T2R16 (SEQ ID NO: 145),T2R38 (SEQ ID NO: 165), T2R39 (SEQ ID NO: 167), T2R40 (SEQ ID NO: 171),T2R41 (SEQ ID NO: 185), T2R44 (SEQ ID NO: 193), T2R46 (SEQ ID NO: 207),T2R47 (SEQ ID NO: 215), T2R48 (SEQ ID NO: 223), T2R49 (SEQ ID NO: 239),T2R50 (SEQ ID NO: 255) and T2R60 (SEQ ID NO: 261).
 5. The array of claim2, which array is a microarray.
 6. A collection comprising two or moreisolated T2R variant-specific nucleic acid molecules, each comprising atleast about 10 contiguous nucleotides spanning at least one T2R SNPposition selected from the group consisting of: position 332 of T2R1(SEQ ID NO: 47); position 616 of T2R1 (SEQ ID NO: 47); position 349 ofT2R3 (SEQ ID NO: 53); position 8 of T2R4 (SEQ ID NO: 61); position 17 ofT2R4 (SEQ ID NO: 61); position 20 of T2R4 (SEQ ID NO: 61); position 186of T2R4 (SEQ ID NO: 61); position 221 of T2R4 (SEQ ID NO: 61); position268 of T2R4 (SEQ ID NO: 61); position 512 of T2R4 (SEQ ID NO: 61);position 77 of T2R5 (SEQ ID NO: 73); position 235 of T2R5 (SEQ ID NO:73); position 338 of T2R5 (SEQ ID NO: 73); position 500 of T2R5 (SEQ IDNO: 73); position 638 of T2R5 (SEQ ID NO: 73); position 881 of T2R5 (SEQID NO: 73); position 254 of T2R7 (SEQ ID NO: 87); position 538 of T2R7(SEQ ID NO: 87); position 640 of T2R7 (SEQ ID NO: 87); position 787 ofT2R7 (SEQ ID NO: 87); position 788 of T2R7 (SEQ ID NO: 87); position 912of T2R7 (SEQ ID NO: 87); position 142 of T2R8 (SEQ ID NO: 97); position370 of T2R8 (SEQ ID NO: 97); position 496 of T2R8 (SEQ ID NO: 97);position 829 of T2R8 (SEQ ID NO: 97); position 922 of T2R8 (SEQ ID NO:97); position 201 of T2R9 (SEQ ID NO: 109); position 381 of T2R9 (SEQ IDNO: 109); position 450 of T2R9 (SEQ ID NO: 109); position 560 of T2R9(SEQ ID NO: 109); position 697 of T2R9 (SEQ ID NO: 109); position 867 ofT2R9 (SEQ ID NO: 109); position 880 of T2R9 (SEQ ID NO: 109); position467 of T2R10 (SEQ ID NO: 131); position 521 of T2R10 (SEQ ID NO: 131);position 691 of T2R10 (SEQ ID NO: 131); position 776 of T2R13 (SEQ IDNO: 133); position 256 of T2R14 (SEQ ID NO: 137); position 589 of T2R14(SEQ ID NO: 137); position 301 of T2R16 (SEQ ID NO: 145); position 481of T2R16 (SEQ ID NO: 145); position 516 of T2R16 (SEQ ID NO: 145);position 665 of T2R16 (SEQ ID NO: 145); position 145 of T2R38 (SEQ IDNO: 165); position 239 of T2R38 (SEQ ID NO: 165); position 785 of T2R38(SEQ ID NO: 165); position 820 of T2R38 (SEQ ID NO: 165); position 886of T9R38 (SEQ ID NO: 165); position 578 of T2R39 (SEQ ID NO: 167);position 589 of T2R39 (SEQ ID NO: 167); position 560 of T2R40 (SEQ IDNO: 171); position 817 of T2R40 (SEQ ID NO: 171); position 871 of T2R40(SEQ ID NO: 171); position 380 of T2R41 (SEQ ID NO: 185); position 584of T2R41 (SEQ ID NO: 185); position 103 of T2R44 (SEQ ID NO: 193;position 484 of T2R44 (SEQ ID NO: 193); position 599 of T2R44 (SEQ IDNO: 193); position 649 of T2R44 (SEQ ID NO: 193); position 656 of T2R44(SEQ ID NO: 193); position 680 of T2R44 (SEQ ID NO: 193); position 718of T2R44 (SEQ ID NO: 193); position 827 of T2R44 (SEQ ID NO: 193);position 843 of T2R44 (SEQ ID NO: 193); position 106 of T2R46 (SEQ IDNO: 207); position 682 of T2R46 (SEQ ID NO: 207); position 749 of T2R46(SEQ ID NO: 207); position 834 of T2R46 (SEQ ID NO: 207); position 862of T2R46 (SEQ ID NO: 207); position 521 of T2R47 (SEQ ID NO: 215);position 577 of T2R47 (SEQ ID NO: 215); position 756 of T2R47 (SEQ IDNO: 215); position 94 of T1R48 (SEQ ID NO: 223); position 113 of T2R48(SEQ ID NO: 223); position 376 of T2R48 (SEQ ID NO: 223); position 456of T2R48 (SEQ ID NO: 223); position 673 of T2R48 (SEQ ID NO: 223);position 719 of T2R48 (SEQ ID NO: 223); position 799 of T2R48 (SEQ IDNO: 223); position 815 of T2R48 (SEQ ID NO: 223); position 895 of T2R48(SEQ ID NO: 223); position 235 of T2R49 (SEQ ID NO: 239); position 421of T2R49 (SEQ ID NO: 239); position 429 of T2R49 (SEQ ID NO: 239);position 442 of T2R49 (SEQ ID NO: 239); position 516 of T2R49 (SEQ IDNO: 239); position 706 of T2R49 (SEQ ID NO: 239); position 755 of T2R49(SEQ ID NO: 239); position 764 of T2R49 (SEQ ID NO: 239); position 808of T2R49 (SEQ ID NO: 239); position 155 of T2R50 (SEQ ID NO: 255);position 181 of T2R50 (SEQ ID NO: 255); position 608 of T2R50 (SEQ IDNO: 255); and position 595 of T2R60 (SEQ ID NO: 261), wherein at leastone of the nucleic acid molecules is a T2R44 variant-specific nucleicacid molecule spanning a SNP selected from T at position 103, C atposition 423, A at position 599, G at position 649, T at position 680, Aat position 718, G at position 744, G at position 827 and A at position843 of SEQ ID NO:
 33. 7. The collection of claim 6, comprising at leastone isolated T2R variant-specific nucleic acid molecule selected fromT2R1, T2R3, T2R4, T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16,T2R38, T2R39, T2R40, T2R41, T2R43, T2R46, T2R47, T2R48, T2R49, T2R50,and T2R60.
 8. The collection of claim 6, comprising at least oneisolated T2R variant-specific nucleic acid molecule from each of:position 332 of T2R1 (SEQ ID NO: 47); position 616 of T2R1 (SEQ ID NO:47); position 349 of T2R3 (SEQ ID NO: 53); position 8 of T2R4 (SEQ IDNO: 61); position 17 of T2R4 (SEQ ID NO: 61); position 20 of T2R4 (SEQID NO: 61); position 186 of T1R4 (SEQ ID NO: 61); position 221 of T2R4(SEQ ID NO: 61); position 268 of T2R4 (SEQ ID NO: 61); position 512 ofT2R4 (SEQ ID NO: 61); position 77 of T2R5 (SEQ ID NO: 73); position 235of T2R5 (SEQ ID NO: 73); position 338 of T2R5 (SEQ ID NO: 73); position500 of T2R5 (SEQ ID NO: 73); position 638 of T2R5 (SEQ ID NO: 73);position 881 of T2R5 (SEQ ID NO: 73); position 254 of T2R7 (SEQ ID NO:87); position 538 of T2R7 (SEQ ID NO: 87); position 640 of T2R7 (SEQ IDNO: 87); position 787 of T2R7 (SEQ ID NO: 87); position 788 of T2R7 (SEQID NO: 87); position 912 of T2R7 (SEQ ID NO: 87); position 142 of T2R8(SEQ ID NO: 97); position 370 of T2R8 (SEQ ID NO: 97); position 496 ofT2R8 (SEQ ID NO: 97); position 829 of T2R8 (SEQ ID NO: 97); position 922of T2R8 (SEQ ID NO: 97); position 201 of T2R9 (SEQ ID NO: 109); position381 of T2R9 (SEQ ID NO: 109); position 450 of T9R9 (SEQ ID NO: 109);position 560 of T2R9 (SEQ ID NO: 109); position 697 of T2R9 (SEQ ID NO:109); position 867 of T2R9 (SEQ ID NO: 109); position 880 of T2R9 (SEQID NO: 109); position 467 of T2R10 (SEQ ID NO: 131); position 521 ofTR10 (SEQ ID NO: 131); position 691 of T9R10 (SEQ ID NO: 131); position776 of T2R13 (SEQ ID NO: 133); position 256 of T2R14 (SEQ ID NO: 137);position 589 of T2R14 (SEQ ID NO: 137); position 301 of T2R16 (SEQ IDNO: 145); position 481 of T2R16 (SEQ ID NO: 145); position 516 of T9R16(SEQ ID NO: 145); position 665 of T2R16 (SEQ ID NO: 145); position 145of T2R38 (SEQ ID NO: 165); position 239 of T2R38 (SEQ ID NO: 165);position 785 of T2R38 (SEQ ID NO: 165); position 820 of T2R38 (SEQ IDNO: 165); position 886 of T2R38 (SEQ ID NO: 165); position 578 of T2R39(SEQ ID NO: 167); position 589 of T2R39 (SEQ ID NO: 167); position 560of T2R40 (SEQ ID NO: 171); position 817 of T2R40 (SEQ ID NO: 171);position 871 of T2R40 (SEQ ID NO: 171); position 380 of T2R41 (SEQ IDNO: 185); position 584 of T2R41 (SEQ ID NO: 185); position 103 of T2R44(SEQ ID NO: 193; position 484 of T2R44 (SEQ ID NO: 193); position 599 ofT2R44 (SEQ ID NO: 193); position 649 of T9R44 (SEQ ID NO: 193); position656 of T2R44 (SEQ ID NO: 193); position 680 of T2R44 (SEQ ID NO: 193);position 718 of T2R44 (SEQ ID NO: 193); position 827 of T2R44 (SEQ IDNO: 193); position 843 of T2R44 (SEQ ID NO: 193); position 106 of T2R46(SEQ ID NO: 207); position 682 of T2R46 (SEQ ID NO: 207); position 749of T2R46 (SEQ ID NO: 207); position 834 of T2R46 (SEQ ID NO: 207);position 862 of T2R46 (SEQ ID NO: 207); position 521 of T2R47 (SEQ IDNO: 215); position 577 of T2R47 (SEQ ID NO: 215); position 756 of T2R47(SEQ ID NO: 215); position 94 of T2R48 (SEQ ID NO: 223); position 113 ofT2R48 (SEQ ID NO: 223); position 376 of T2R48 (SEQ ID NO: 223); position456 of T2R48 (SEQ ID NO: 223); position 673 of T2R48 (SEQ ID NO: 223);position 719 of T2R48 (SEQ ID NO: 223); position 799 of T2R48 (SEQ IDNO: 223); position 815 of T2R48 (SEQ ID NO: 223); position 895 of T2R48(SEQ ID NO: 223); position 235 of T2R49 (SEQ ID NO: 239); position 421of T2R49 (SEQ ID NO: 239); position 499 of T2R49 (SEQ ID NO: 239);position 442 of T2R49 (SEQ ID NO: 239); position 516 of T2R49 (SEQ IDNO: 239); position 706 of T2R49 (SEQ ID NO: 239); position 755 of T2R49(SEQ ID NO: 239); position 764 of T2R49 (SEQ ID NO: 239); position 808of T2R49 (SEQ ID NO: 239); position 155 of T2R50 (SEQ ID NO: 255);position 181 of T2R50 (SEQ ID NO: 255); position 608 of T9R50 (SEQ IDNO: 255); and position 595 of T2R60 (SEQ ID NO. 261).
 9. The collectionof claim 6, further comprising at least one isolated T2Rvariant-specific nucleic acid molecule from each of SEQ ID NO: 49, 55,57, 59, 63, 65, 67, 69, 71, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 99,101, 103, 105, 107, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,135, 139, 141, 147, 149, 151, 155, 157, 161, 163, 169, 173, 175, 177,179, 181, 183, 187, 189, 191, 197, 199, 201, 203, 205, 209, 211, 213,217, 219, 225, 227, 229, 231, 233, 235, 237, 241, 243, 245, 247, 249,251, 253, 257, 259, and
 263. 10. The collection of claim 6, wherein eachnucleic acid molecule is stored in a separate container.
 11. Thecollection of claim 10, wherein the separate containers are wells of amicrotiter plate or equivalent thereof.
 12. The collection of claim 6,wherein the nucleic acid molecules of the collection are affixed to asolid surface in an array.
 13. The collection of claim 12, wherein thearray is a microarray.
 14. The collection of claim 13, which comprisesnucleic acid molecules having the sequence as set forth in SEQ ID NO:47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,255, 257, 259, 261, or
 263. 15. The collection of claim 6, wherein theisolated T2R variant-specific nucleic acid molecules comprise: (a) SEQID NOs: 47, 49, and 51; (b) SEQ ID NOs: 53 and 55; (c) SEQ ID NOs: 57,59, 61, 63, 65, 67, 69, and 71; (d) SEQ ID NOs: 73, 75, 77, 79, 81, 83,and 85; (e) SEQ ID NOs: 87, 89, 91, 93, and 95; (f) SEQ ID NOs: 97, 99,101, 103, 105, and 107; (g) SEQ ID NOs: 109, 111, 113, 115, 117, 119,121, and 123; (h) SEQ ID NOs: 125, 127, 129, and 131; (i) SEQ ID NOs:133 and 135; (j) SEQ ID NOs: 137, 139, and 141; (k) SEQ ID NOs: 143,145, 147, 149, and 151; (l) SEQ ID NOs: 153, 155, 157, 159, 161, 163,and 165; (m) SEQ ID NOs: 167 and 169; (n) SEQ ID NOs: 171, 173, 175, and179; (o) SEQ ID NOs: 181, 183, and 185; (p) SEQ ID NOs: 187, 189, 191,193, 195, 197, and 199; (q) SEQ ID NOs: 201, 203, 205, 207, 209, and211; (r) SEQ ID NOs: 213, 215, 217, and 219; (s) SEQ ID NOs: 221, 223,225, 227, 229, 231, 233, 235, and 237; (t) SEQ ID NOs: 239, 241, 243,245, 247, 249 and 251; (u) SEQ ID NOs: 253, 255, 257, and 259; (v) SEQID NOs: 261 and 263; or (w) a combination of two or more of (a) through(v).
 16. The collection of claim 6, comprising at least one isolated T2Rvariant-specific nucleic acid molecule from each of T2R1, T2R3, T2R4,T2R5, T2R7, T2R8, T2R9, T2R10, T2R13, T2R14, T2R16, T2R38, T2R39, T2R40,T2R41, T2R43, T2R46, T2R47, T2R48, T2R49, T2R50, and T2R60.
 17. Anisolated nucleic acid molecule, encoding a T2R44 polypeptide isoformcomprising the amino acid sequence selected from SEQ ID NO: 188, 190,192, 198 and
 200. 18. A vector comprising the isolated nucleic acidmolecule of claim
 17. 19. An isolated host cell comprising the vector ofclaim
 18. 20. An isolated nucleic acid molecule comprising a nucleotidesequence encoding a T2R44 allele, wherein the nucleotide sequence isselected from SEQ ID NO: 187, 189, 191, 197, and
 199. 21. A vectorcomprising the isolated nucleic acid molecule of claim
 20. 22. Anisolated host cell comprising the vector of claim
 21. 23. A method ofdetermining a T2R44 genotype of a subject, comprising: obtaining a testsample of DNA containing a T2R44 sequence of the subject; and detectinga polymorphism in the T2R44 sequence by contacting the test sample withthe nucleic acid molecule of claim 1, wherein the polymorphism isselected from T at position 103, C at position 423, A at position 599, Gat position 649, T at position 680, A at position 718, G at position744, G at position 827 and A at position 843 of SEQ ID NO:
 33. 24. A kitfor determining whether or not a subject has a selected T2R44 genotypeor haplotype, comprising: a container comprising at least oneoligonucleotide specific for a T2R44 sequence comprising at least “about10 contiguous nucleotides spanning at least”, one single nucleotidepolymorphism selected from T at position 103, C at position 423, A atposition 599, G at position 649, T at position 680, A at position 718, Gat position 744, G at position 827 and A at position 843 of SEQ ID NO:33; and instructions for using the kit, the instructions indicatingsteps for: performing a method to detect the presence of variant T2R44nucleic acid in the sample; and analyzing data generated by the method,wherein the instructions indicate that the presence of the variantnucleic acid in the sample indicates that the individual has theselected T2R44 genotype or haplotype.
 25. The kit of claim 24, furthercomprising a container that comprises a detectable oligonucleotide.